Compositions and methods for the treatment of neurodegenerative and other diseases

ABSTRACT

In one embodiment, the present application discloses methods of treating diseases and disorders with sulfasalazine and pharmaceutical formulations of sulfasalazine where the bioavailability of the sulfasalazine is increased. In another embodiment, the present application also provides dosing regimens for treating neurodegenerative diseases and disorders with compositions comprising sulfasalazine.

This application is a Continuation application of U.S. Non-Provisionalapplication Ser. No. 15/365,843 filed Nov. 30, 3016, which is aContinuation application of U.S. Non-Provisional application Ser. No.14/170,405 filed Jan. 31, 2014 issued as U.S. Pat. No. 9,597,339 on Mar.21, 2017, which claims the benefit of U.S. Provisional Application No.61/759,933 filed Feb. 1, 2013 and U.S. Provisional Application No.61/780,340 filed Mar. 13, 2013, all of which are incorporated herein byreference.

FIELD OF THE INVENTION

In one embodiment, the present invention relates to methods of treatingdiseases and disorders with sulfasalazine and pharmaceuticalformulations of sulfasalazine where the bioavailability of thesulfasalazine is increased. In another embodiment, the present inventionalso provides dosing regimens for treating neurodegenerative diseases ordisorders, such as P-MS and ALS with compositions comprisingsulfasalazine.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases are collectively a leading cause of death anddisability. Examples of neurodegenerative diseases include progressivemultiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease,Parkinson's disease, epilepsy, neuropathic pain, Huntington's diseaseand traumatic brain injury. While the ultimate causes and naturalhistories of the individual neurodegenerative diseases differ, commonpathological processes occur in most, if not all, neurodegenerativediseases. These common pathological processes include high levels ofactivated glial cells (“neuroinflammation”), dysregulated glutamatesignaling and chronic damage to axons and neurons.

Progressive multiple sclerosis (P-MS) is a devastating neurodegenerativedisease that affects approximately 120,000 people in the United Statesand 350,000 people in the developed world. P-MS patients progressivelyaccumulate disabilities, including changes in sensation (hypoesthesia),muscle weakness, abnormal muscle spasms, or difficulty moving;difficulties with coordination and balance; problems in speech(dysarthria) or swallowing (dysphagia), visual problems (nystagmus,optic neuritis, phosphenes or diplopia), fatigue and acute or chronicpain syndromes, bladder and bowel difficulties, cognitive impairment, oremotional symptomatology (mainly major depression). The only drugcurrently approved to treat P-MS in the United States is mitoxantrone(Novantrone), a cytotoxic agent that is also used to treat cancers.Mitoxantrone has a serious adverse effect profile and carries a lifetimelimit on exposure. The treatment of P-MS remains a significant unmetmedical need.

There are three major sub-types of P-MS recognized by the NationalMultiple Sclerosis Society (US): Primary Progressive Multiple Sclerosis(PP-MS), Secondary Progressive Multiple Sclerosis (SP-MS) andProgressive-Relapsing Multiple Sclerosis (PR-MS). Approximately 85% ofmultiple sclerosis patients clinically present with Relapse RemittingMultiple Sclerosis (RR-MS), characterized by episodes of acuteneurological deficits (relapses), followed by partial or completerecovery of the deficits. After a median time to conversion of around 19years, approximately 70% of RR-MS patients develop a progressiveneurological decline, clinically recognized as SP-MS. Approximately 10%of multiple sclerosis patients clinically present with PP-MS,characterized by a progressive neurological decline with few to nopreceding episodes of neurological deficits (relapses), while 5% presentwith PR-MS, characterized by a steady worsening disease from the onsetbut also have clear acute flare-ups (relapses), with or withoutrecovery, e.g. Compton et al, Lancet 372:1502-1517 (2008); Trapp et al,Annu. Rev. Neurosci. 31:247-269 (2008). Here, PP-MS, SP-MS and PR-MS aregrouped together as P-MS, as they share many similarities, includingnatural history, clinical manifestations and pathology, e.g.Kremenchutsky et al, Brain 129:584-594 (2006); Lassmann et al, Nat. Rev.Neurology 8:647-656 (2012); Stys et al, Nat. Rev. Neuroscience13:507-514 (2012).

Thus far, drugs that are effective for RR-MS have not shown efficacy inP-MS, e.g. Fox et al, Multiple Sclerosis Journal 18:1534-1540 (2012).This is believed to be due to current RR-MS drugs primarily targetingthe peripheral immune system (B and T-cells) while P-MS is insteaddriven by resident CNS inflammatory cells, including microglia andastrocytes, e.g. Fitzner et al, Curr. Neuropharmacology 8:305-315(2008); Weiner, J. Neurology 255, Suppl. 1:3-11 (2008); LassmanNeurology 8:647-656 (2012). Recent evidence suggests that the efficacyof mitoxantrone in P-MS may be due to inhibition of activation ofastrocytes, thereby linking anti-neuroinflammation with efficacy inP-MS, e.g. Burns et al, Brain Res. 1473: 236-241 (2012).

In addition to resident CNS neuroinflammation, P-MS is also accompaniedby the loss of axons and ultimately death of neuronal cells. Themechanisms that drive axonal and neuronal damage are not completelyunderstood, although glutamate excitotoxicity is one of the leadingsuspects in human P-MS, e.g. Frigo, Curr. Medicin. Chem. 19:1295-1299(2012). In particular, oligodendrocytes are especially sensitive toelevated levels of glutamate, e.g. Matute, J. Anatomy 219:53-64 (2011).Subsets of MS patients have been demonstrated to have elevatedextracellular glutamate levels in the cerebrospinal fluid, e.g.Sarchielli et al, Arch. Neurol. 60:1082-1088 (2003) and P-MS patientshave an increased incidence of seizures and neuropathic pain; bothconditions may derive from excessive glutamate signaling and are treatedclinically with anti-glutamatergics, e.g. Eriksson et al, Mult. Scler.8:495-499 (2002); Svendsen et al, Pain 114: 473-481 (2004).

Another neurodegenerative disease thought to involve excessiveglutamatergic signaling is amyotrophic lateral sclerosis (ALS), whichaffects approximately 100,000 patients in the developed world. ALSpatients progressively lose motor neuron function, causing muscularatrophy, paralysis and death. The average lifespan after diagnosis isonly 3-5 years. Riluzole (Rilutek) is the only known treatment that hasbeen found to improve survival in ALS patients; however, the treatmentis effective only to a modest extent by lengthening the survival time byonly several months. Thus treatment of ALS remains a significant unmetmedical need.

At the molecular level, ALS is characterized by excessive glutamatergicsignaling leading to neuroexcitotoxicity and motor neuron death; see,e.g. Bogaert et al, CNS Neurol. Disord. Drug Targets 9:297-304 (2010).Affected tissues in the spinal cord also have high levels of activatedmicroglia and activated astrocytes, collectively recognized asneuroinflammation; see, e.g. Philips et al, Lancet Neurol. 10:253-263(2011) and neuroinflammatory cells have been shown to drive diseaseprogression in ALS animal models; see, e.g. Ilieva et al, J. Cell Biol.187: 761-772 (2009). The glutamate pathway has been clinically validatedin ALS, as Riluzole inhibits multiple glutamate activities, includingthe activity of AMPA glutamate receptor; see, e.g., Lin et al,Pharmacology 85:54-62 (2010).

Approximately 10% of ALS cases are familial, while the remainders arebelieved to be sporadic, with no clear genetic cause to date. Among thefamilial cases, approximately 20% are due to mutations in the SOD1 gene.Mice and rats genetically altered to contain the mutant human SOD1 genedevelop motor neuron disease that phenotypically resembles human ALS.Because of this, most potential ALS therapies are tested in the SOD1mouse or rat model for efficacy.

Excessive glutamatergic signaling is believed to play a causal role inneurodegenerative diseases besides P-MS and ALS. For instance,neuropathic pain is a chronic condition caused by damage or disease thataffects the somatosensory system. Neuropathic pain is associated withneuronal hyperexcitability, a common consequence of excessive glutamatesignaling, see, e.g. Baron et al, Lancet Neurology 9: 807-819 (2010).Neuropathic pain may manifestin abnormal sensations called dysesthesiaand pain produced by normally non-painful stimuli (allodynia).Neuropathic pain may have continuous and/or episodic (paroxysmal)components. The latter are likened to an electric shock. Commonqualities include burning or coldness, “pins and needles” sensations,numbness and itching. Neuropathic pain is clinically treated withcompounds that possess anti-glutamatergic activity (e.g. Topamax,Pregabalin). Importantly, sulfasalazine has previously been shown tohave efficacy in models of diabetic neuropathy (e.g. Berti-Mattera etal, Diabetes 57: 2801-2808 (2008); U.S. Pat. No. 7,964,585) andcancer-induced bone pain (e.g. Ungard et al, Pain 155: 28-36 (2014)),and is currently being evaluated in clinical trials of painful diabeticneuropathy (see Massachusetts General Hospital, Clinical TrialsIdentifier NCT01667029).

Other neurodegenerative diseases where compounds with anti-glutamatergicactivity are used clinically include Parkinson's disease (Amantadine andBudipine), Alzheimer's disease (Memantine), and epilepsy (Carbamazepine,Lamictal, and Keppra). Anti-glutamatergics are being investigated fortreatment of traumatic brain injury, Huntington's disease, multiplesclerosis, and ischemic stroke. In many cases, these neurologicaldiseases are also accompanied by high levels of neuroinflammation. Otherneurological diseases that are linked to excessive glutamate signalingand neuroinflammation include Rett Syndrome, Frontotemporal Dementia,HIV-associated Dementia, Tuberous Sclerosis and Alexander disease.

The system x_(c) ⁻ glutamate-cysteine exchange transporter (herein“system x_(c) ⁻”) is the only glutamate transporter that normallyfunctions to release glutamate into the extracellular space. The amountof glutamate released by system x_(c) ⁻ is sufficient to stimulatemultiple ionotropic and metabotropic glutamate receptors in vivo.Current anti-glutamatergics target either the vesicular release ofglutamate or individual glutamate receptors that lie downstream ofglutamate release (e.g., riluzole to the AMPA receptor). In contrast,system x_(c) ⁻ is responsible for the non-vesicular release of glutamateand lies upstream of the individual glutamate receptors. The protein xCT(SLC7A11) is the only currently identified catalytic component of systemx_(c) ⁻.

Sulfasalazine (also referred to as 2-hydroxy-5-[(E)-2-{4-[(pyridin-2-yl)sulfamoyl] phenyl}diazen-1-yl]benzoic acid, 5-([p(2-pyridylsulfamoyl)phenyl]azo) salicylic acid or salicylazosulfapyridine) is a conjugate of5-aminosalicylate and sulfapyridine, and is widely prescribed forinflammatory bowel disease, rheumatoid arthritis, and ankylosingspondylitis. Sulfasalazine is degraded by intestinal bacteria into itsmetabolites, 5-aminosalicylate and sulfapyridine. The mechanism ofaction in inflammatory bowel disease and rheumatoid arthritis isunknown, although action in the colon may be mediated by a metabolite,5-aminosalicylate. Sulfasalazine has been shown to be an inhibitor ofsystem x_(c) ⁻.

The current U.S. on-market formulations of sulfasalazine (e.g.Azulfidine®) have poor bioavailability, with only approximately 15% ofthe compound reaching the circulation following oral dosing (see, forexample, Label for Azulfidine®sulfasalazine tablets, USP). A majortoxicity concern is exposure of the gastrointestinal tract tosulfasalazine, where it causes nausea, diarrhea and cramping in adose-dependent manner, see e.g. Weaver, J. Clin. Rheumatol. 5: 193-200(1999). An additional toxicity concern is sulfapyridine, one of themetabolites of sulfasalazine. Sulfapyridine is highly (>70%)bioavailable and is believed to be produced by intestinal bacteria, see,e.g., Peppercorn, M., J. Chin. Pharmacol. 27: 260-265 (1987); Watkinson,G., Drugs 32: Suppl 1:1-11 (1986).

SUMMARY OF THE INVENTION

The present application provides methods targeting system x_(c) ⁻ as atherapeutic approach to diseases involving excessive glutamatergicsignaling. The present application describes administering an inhibitorof system x_(c) ⁻, such as sulfasalazine, to treat neurodegenerativediseases involving excessive glutamatergic signaling, such as P-MS andALS. Without being bound by any theory asserted herein, the workinghypothesis is that system x_(c) ⁻, by releasing excessive amounts ofglutamate, causes neuronal damage, thereby activating neuroinflammatorycells. This in turn elevates levels of system x_(c) ⁻, causing apositive feedback loop that damages and ultimately kills axons andneurons, including motor neurons. Inhibiting system x_(c) ⁻ with aninhibitor such as sulfasalazine can interrupt this feedback loop and canreduce damage to the axons and neurons, including motor neurons.

Thus, the present application provides methods of treatment of variousdiseases using system x_(c) ⁻ inhibitors, including methods usingimproved dosing regimens. In addition, the present application describesformulations of a system x_(c) ⁻ inhibitor, sulfasalazine, where thoseformulations increase the bioavailability of orally-administeredsulfasalazine. Those formulations can be used in the treatment ofneurodegenerative diseases and disorders as well as other diseases anddisorders, including rheumatoid arthritis and ankylosing spondylitis(diseases for which sulfasalazine is currently approved in variousmarkets).

Experiments described herein using a mouse model of neurodegenerationdemonstrate that treatment with sulfasalazine significantly reduceslevels of neuroinflammatory cells in the spinal cord (see Example 3),including both activated microglia and activated astrocytes. Inaddition, experiments described herein using a mouse model of ALSdemonstrate that the administration of sulfasalazine increases absolutesurvival and the survival after onset of definitive neurological diseasein the SOD1 mouse model of ALS (see Example 1). Thus, in variousembodiments, the present invention provides methods for treating P-MS,ALS, and other neurodegenerative diseases by administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient. In certain embodiments, methods are provided for treatingother neurodegenerative diseases involving excessive glutamatergicsignaling comprising administering to the patient with such aneurodegenerative disease a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the neurodegenerative disease is selectedfrom Parkinson's disease, Alzheimer's disease, epilepsy, neuropathicpain, traumatic brain injury, Huntington's disease, ischemic stroke,Rett Syndrome, Frontotemporal Dementia, HIV-associated Dementia,Tuberous Sclerosis and Alexander disease.

Increasing the Bioavailability of Sulfasalazine

One challenge with treating P-MS, ALS and other diseases withpharmaceutical compositions comprising sulfasalazine is the poor oralbioavailability of the standard formulations of sulfasalazine. Forexample, only 15% of the sulfasalazine in an orally administered dose ofAzulfidine is absorbed into the bloodstream (see AzulfidineSulfasalazine Tablets Label, LAB-0241-3.0, revised October 2009).Because the level of sulfasalazine at the sites of action relevant toneurodegenerative diseases (such as the spinal cord) is proportional tothe amount of sulfasalazine in the plasma (see Example 4), the poorbioavailability of the current oral formulation of sulfasalazine limitsthe amount of sulfasalazine that reaches such sites of action. Thus, useof a standard formulation of sulfasalazine to treat neurodegenerativediseases would require large oral doses of sulfasalazine to beadministered. This would expose patients to high levels of sulfasalazinein the gastrointestinal tract and generate high levels of sulfapyridinein the plasma, thereby increasing toxicity. One of the aims of thisinvention is to address these issues by improving the oralbioavailability of sulfasalazine for the treatment of P-MS, ALS or otherdiseases, including non-neurodegenerative diseases. Increasing suchbioavailability would allow dosing levels of sulfasalazine to be lower,with the further benefit of limiting gastrointestinal exposure tosulfasalazine and systemic exposure to sulfapyridine. The formulationsprovided herein are predicted to increase the therapeutic index forsulfasalazine in the treatment of various diseases.

The present invention provides various methods of treating variousdiseases and disorders using the compositions described herein in whichthe solubility and/or bioavailability of sulfasalazine has beenincreased. Such methods are described below.

In certain embodiments, there are provided methods for treating adisease or disorder in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat oral administration of the formulated pharmaceutical composition toa rat results in a plasma level of sulfasalazine thirty minutes aftersuch administration that is at least 25%, at least 50%, at least 100%,at least 150%, at least 200%, at least 250% or at least 300% higher thanthe plasma level of sulfasalazine thirty minutes after administration ofthe same dose level of crystalline sulfasalazine to a rat as determinedby the method of Example 10. In certain embodiments, there are providedmethods for treating a disease or disorder in a patient comprisingorally administering to the patient a pharmaceutical compositioncomprising a therapeutically effective amount of sulfasalazine and apharmaceutically acceptable excipient, wherein the pharmaceuticalcomposition is formulated such that oral administration of theformulated pharmaceutical composition to a rat results in a plasma levelof sulfasalazine thirty minutes after such administration that is about25%, about 50%, about 100%, about 150%, about 200%, about 250% or about300% higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In yet other embodiments,there are provided methods for treating a disease or disorder in apatient comprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is between about 25% and about 500%, inclusive, higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In certain of those embodiments, thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is between about 75% and about 300%, inclusive, higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In certain other of the embodiments, thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is between about 100% and about 200%, inclusive, higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In certain of the embodiments described inthis paragraph, the disease or disorder is a neurodegenerative diseaseor disorder such as P-MS or ALS. In certain of the embodiments describedin this paragraph, the disease or disorder is other than aneurodegenerative disease or disorder. In certain of those embodiments,the disease or disorder is rheumatoid arthritis or ankylosingspondylitis. In certain embodiments described in this paragraph, thepharmaceutical composition is one of the pharmaceutical compositionsprovided by the present application. In certain of those embodiments,the pharmaceutical composition is in an oral dosage form or in a spraydried dispersion form.

In yet other embodiments, there are provided methods for treating P-MSin a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is at least 25%, at least 50%, at least 100%, atleast 150%, at least 200%, at least 250% or at least 300% higher thanthe plasma level of sulfasalazine thirty minutes after administration ofthe same dose level of crystalline sulfasalazine to a rat as determinedby the method of Example 10. In certain embodiments, there are providedmethods for treating P-MS in a patient comprising orally administeringto the patient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat oral administration of the formulated pharmaceutical composition toa rat results in a plasma level of sulfasalazine thirty minutes aftersuch administration that is about 25%, about 50%, about 100%, about150%, about 200%, about 250% or about 300% higher than the plasma levelof sulfasalazine thirty minutes after administration of the same doselevel of crystalline sulfasalazine to a rat as determined by the methodof Example 10. In yet other embodiments, there are provided methods fortreating P-MS in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat oral administration of the formulated pharmaceutical composition toa rat results in a plasma level of sulfasalazine thirty minutes aftersuch administration that is between about 25% and about 500%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain of thoseembodiments, the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 75% and about 300%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain of thoseembodiments, the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 100% and about 200%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In yet other embodiments, there are provided methods for treating ALS ina patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is at least 25%, at least 50%, at least 100%, atleast 150%, at least 200%, at least 250% or at least 300% higher thanthe plasma level of sulfasalazine thirty minutes after administration ofthe same dose level of crystalline sulfasalazine to a rat as determinedby the method of Example 10. In certain embodiments, there are providedmethods for treating ALS in a patient comprising orally administering tothe patient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat oral administration of the formulated pharmaceutical composition toa rat results in a plasma level of sulfasalazine thirty minutes aftersuch administration that is about 25%, about 50%, about 100%, about150%, about 200%, about 250% or about 300% higher than the plasma levelof sulfasalazine thirty minutes after administration of the same doselevel of crystalline sulfasalazine to a rat as determined by the methodof Example 10. In yet other embodiments, there are provided methods fortreating ALS in a patient comprising orally administering to the patienta pharmaceutical composition comprising a therapeutically effectiveamount of sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 25% and about 500%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain of thoseembodiments, the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 75% and about 300%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain of thoseembodiments, the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 100% and about 200%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In yet other embodiments, there are provided methods for treatingneuropathic pain in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat oral administration of the formulated pharmaceutical composition toa rat results in a plasma level of sulfasalazine thirty minutes aftersuch administration that is at least 25%, at least 50%, at least 100%,at least 150%, at least 200%, at least 250% or at least 300% higher thanthe plasma level of sulfasalazine thirty minutes after administration ofthe same dose level of crystalline sulfasalazine to a rat as determinedby the method of Example 10. In certain embodiments, there are providedmethods for treating neuropathic pain in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that oral administration of the formulatedpharmaceutical composition to a rat results in a plasma level ofsulfasalazine thirty minutes after such administration that is about25%, about 50%, about 100%, about 150%, about 200%, about 250% or about300% higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In yet other embodiments,there are provided methods for treating neuropathic pain in a patientcomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is between about 25% and about 500%, inclusive, higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In certain of those embodiments, thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is between about 75% and about 300%, inclusive, higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In certain of those embodiments, thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is between about 100% and about 200%, inclusive, higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In some of the embodiments in this paragraph,the neuropathic pain results from painful diabetic neuropathy. Incertain embodiments, the neuropathic pain manifests as dysesthesia. Incertain embodiments, the neuropathic pain manifests as allodynia. Incertain embodiments described in this paragraph, the pharmaceuticalcomposition is one of the pharmaceutical compositions provided by thepresent application. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform.

Formulations of the Invention

The present invention provides various pharmaceutical compositionscomprising sulfasalazine that have been formulated to increase thebioavailability of the sulfasalazine. One way to increase thebioavailability of an orally-administered poorly soluble drug such assulfasalazine is to increase the solubility of the drug. The inventionprovides various reformulations of sulfasalazine that increase thesolubility of sulfasalazine (for three non-limiting examples, seeExample 6 herein).

In some embodiments, there is provided a pharmaceutical compositioncomprising sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition has been formulated such that thein vitro solubility of the sulfasalazine is at least 2300 μg/ml at a pHof 5.5 determined as in Example 9. For the purposes of this application,the “in vitro solubility” of sulfasalazine will be considered to be theC_(max) IB at 90 minutes as shown in Example 9 and Table 9 of thisapplication. In other embodiments, there is provided a pharmaceuticalcomposition comprising sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition has been formulatedsuch that the in vitro solubility of the sulfasalazine is at least 500μg/ml or at least 1200 μg/ml at a pH of 5.5 determined as in Example 9.In some embodiments, there is provided a pharmaceutical compositioncomprising sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition has been formulated such that thein vitro solubility of the sulfasalazine is about 500 μg/ml, about 1200μg/ml, or about 2300 μg/ml at a pH of 5.5 determined as in Example 9. Insome embodiments, there is provided a pharmaceutical compositioncomprising sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition has been formulated such that thein vitro solubility of the sulfasalazine is between about 500 μg/ml andabout 11,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example9. In another embodiment of the composition, the in vitro solubility ofthe sulfasalazine is between about 500 μg/ml and about 7,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In yet anotherembodiment of the composition, the in vitro solubility of thesulfasalazine is between about 500 μg/ml and about 5,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In certain ofthose embodiments, the pharmaceutical composition has been formulatedsuch that the in vitro solubility of the sulfasalazine is between about500 μg/ml and about 2500 μg/ml, inclusive, at a pH of 5.5 determined asin Example 9. In certain of those embodiments, the pharmaceuticalcomposition has been formulated such that the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In certainembodiments in this paragraph, the sulfasalazine is in an essentiallyamorphous form.

In some embodiments, there is provided an oral dosage form forsulfasalazine comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein theoral dosage form has been formulated such that the in vitro solubilityof the sulfasalazine is at least 2300 μg/ml at a pH of 5.5 determined asin Example 9. In other embodiments, there is provided an oral dosageform for sulfasalazine comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein theoral dosage form has been formulated such that the in vitro solubilityof the sulfasalazine is at least 500 μg/ml or at least 1200 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In someembodiments, there is provided an oral dosage form for sulfasalazinecomprising a therapeutically effective amount of sulfasalazine and apharmaceutically acceptable excipient, wherein the oral dosage form hasbeen formulated such that the in vitro solubility of the sulfasalazineis about 500 μg/ml, about 1200 μg/ml, or about 2300 μg/ml at a pH of 5.5determined as in Example 9. In some embodiments, there is provided anoral dosage form for sulfasalazine comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the oral dosage form has been formulated such thatthe in vitro solubility of the sulfasalazine is between about 500 μg/mland about 11,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9. In certain other embodiments, the oral dosage form has beenformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 2500 μg/ml at a pH of 5.5 determinedas in Example 9. In certain of those embodiments, the oral dosage formhas been formulated such that the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In anotherembodiment, the oral dosage form has been formulated such that the invitro solubility of the sulfasalazine is between about 2300 μg/ml andabout 7,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9.In another embodiment, the oral dosage form has been formulated suchthat the in vitro solubility of the sulfasalazine is between about 2300μg/ml and about 5,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9. In certain embodiments in this paragraph, the sulfasalazineis in an essentially amorphous form.

In some embodiments, there is provided a pharmaceutical compositioncomprising sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition has been formulated such that thein vitro solubility of the sulfasalazine at a pH of 5.5 as determined asin Example 9 is at least 2 times higher than the in vitro solubility ofcrystalline sulfasalazine in aqueous solution at a pH of 5.5. In certainof those embodiments, the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine at a pH of 5.5 asdetermined in Example 9 is at least 5 times or at least 8.8 times higherthan the in vitro solubility of crystalline sulfasalazine at a pH of 5.5by AUC analysis. In some embodiments, there is provided a pharmaceuticalcomposition comprising sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine at a pH of 5.5 asdetermined as in Example 9 is about 2 times, about 5 times, or about 8.8times higher than the in vitro solubility of crystalline sulfasalazineat a pH of 5.5 by AUC analysis. In some embodiments, there is provided apharmaceutical composition comprising sulfasalazine and apharmaceutically acceptable excipient, wherein the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is betweenabout 2 times and about 44 times, inclusive, higher than the in vitrosolubility of crystalline sulfasalazine at a pH of 5.5 by AUC analysis.In certain of those embodiments, pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined as in Example 9 is between about 2 times andabout 8.8 times, inclusive, higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5 by AUC analysis. In certainother embodiments, pharmaceutical composition is formulated such thatthe in vitro solubility of the sulfasalazine at a pH of 5.5 asdetermined as in Example 9 is between about 8.8 times and about 44times, inclusive, higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain embodiments inthis paragraph, the sulfasalazine is in an essentially amorphous form.

In some embodiments, there is provided an oral dosage form forsulfasalazine comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein theoral dosage form is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is at least 2times higher than the in vitro solubility of crystalline sulfasalazineat a pH of 5.5. In certain of those embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined in Example 9 is at least 5times or at least 8.8 times higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5 by AUC analysis. In someembodiments, there is provided an oral dosage form for sulfasalazinecomprising a therapeutically effective amount of sulfasalazine and apharmaceutically acceptable excipient, wherein the oral dosage form isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined as in Example 9 is about 2 times, about 5 times,or about 8.8 times higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In some embodiments, thereis provided an oral dosage form for sulfasalazine comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the oral dosage form is formulated suchthat the in vitro solubility of the sulfasalazine at a pH of 5.5 asdetermined as in Example 9 is between about 2 times and about 44 times,inclusive, higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain of thoseembodiments, the oral dosage form is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined as inExample 9 is between about 2 times and about 8.8 times, inclusive,higher than the in vitro solubility of crystalline sulfasalazine at a pHof 5.5 by AUC analysis. In certain other embodiments, the oral dosageform is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is betweenabout 8.8 times and about 44 times, inclusive, higher than the in vitrosolubility of crystalline sulfasalazine at a pH of 5.5 by AUC analysis.In certain embodiments in this paragraph, the sulfasalazine is in anessentially amorphous form.

In some embodiments, there is provided a pharmaceutical compositioncomprising sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition has been formulated such thatoral administration of such formulated pharmaceutical composition to arat results in a plasma level of sulfasalazine thirty minutes after suchadministration that is at least 25%, at least 50%, at least 100%, atleast 150%, at least 200%, at least 250%, or at least 300% higher thanthe plasma level of sulfasalazine thirty minutes after administration ofthe same dose level of crystalline sulfasalazine to a rat as determinedin Example 10. In some embodiments, there is provided a pharmaceuticalcomposition comprising sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition has been formulatedsuch that oral administration of the formulated pharmaceuticalcomposition to a rat results in a plasma level of sulfasalazine thirtyminutes after such administration that is about 25%, about 50%, about100%, about 150%, about 200%, about 250% or about 300% higher than theplasma level of sulfasalazine thirty minutes after administration of thesame dose level of crystalline sulfasalazine to a rat as determined bythe method of Example 10. In some embodiments, there is provided apharmaceutical composition comprising sulfasalazine and apharmaceutically acceptable excipient, wherein the pharmaceuticalcomposition has been formulated such that oral administration of suchformulated pharmaceutical composition to a rat results in a plasma levelof sulfasalazine thirty minutes after such administration that isbetween about 25% and about 500%, inclusive, higher than the plasmalevel of sulfasalazine thirty minutes after administration of the samedose level of crystalline sulfasalazine to a rat as determined inExample 10. In certain of those embodiments, the pharmaceuticalcomposition has been formulated such that oral administration of suchformulated pharmaceutical composition to a rat results in a plasma levelof sulfasalazine thirty minutes after such administration that isbetween about 75% and about 300%, inclusive, higher than the plasmalevel of sulfasalazine thirty minutes after administration of the samedose level of crystalline sulfasalazine to a rat as determined inExample 10. In certain of those embodiments, the pharmaceuticalcomposition has been formulated such that oral administration of suchformulated pharmaceutical composition to a rat results in a plasma levelof sulfasalazine thirty minutes after such administration that isbetween about 300% and about 500%, inclusive, higher than the plasmalevel of sulfasalazine thirty minutes after administration of the samedose level of crystalline sulfasalazine to a rat as determined inExample 10. In certain embodiments in this paragraph, the sulfasalazineis in an essentially amorphous form.

In some embodiments, there is provided an oral dosage form forsulfasalazine comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein theoral dosage form is formulated such that oral administration of suchformulated oral dosage form to a rat results in a plasma level ofsulfasalazine thirty minutes after such administration that is at least25%, at least 50%, at least 100%, at least 150%, at least 200%, at least250%, or at least 300% higher than the plasma level of sulfasalazinethirty minutes after administration of the same dose level ofcrystalline sulfasalazine to a rat as determined in Example 10. In someembodiments, there is provided an oral dosage form for sulfasalazinecomprising a therapeutically effective amount of sulfasalazine and apharmaceutically acceptable excipient, wherein the oral dosage form isformulated such that oral administration of such formulated oral dosageform to a rat results in a plasma level of sulfasalazine thirty minutesafter such administration that is between about 25% and about 500%,inclusive, higher than the plasma level of sulfasalazine thirty minutesafter administration of the same dose level of crystalline sulfasalazineto a rat as determined in Example 10. In certain of those embodiments,the oral dosage form is formulated such that oral administration of suchformulated oral dosage form to a rat results in a plasma level ofsulfasalazine thirty minutes after such administration that is betweenabout 75% and about 300%, inclusive, higher than the plasma level ofsulfasalazine thirty minutes after administration of the same dose levelof crystalline sulfasalazine to a rat as determined in Example 10. Incertain of those embodiments, the oral dosage form is formulated suchthat oral administration of such formulated oral dosage form to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 300% and about 500% higher,inclusive, than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined in Example 10. In certain embodiments in thisparagraph, the sulfasalazine is in an essentially amorphous form.

In certain embodiments of the compositions and methods described in thisapplication, the composition comprises a pharmaceutically acceptablepolymer. In certain embodiments, the pharmaceutically acceptable polymermay be selected from polyvinylpyrrolidone (PVP, including PVP VA64,homo- and copolymers of polyvinylpyrrolidone and homopolymers orcopolymers of N-vinylpyrrolidone); crospovidone;polyoxyethylene-polyoxypropylene copolymers (also known as poloxamers);cellulose derivatives (including hydroxypropyl methyl cellulose acetatesuccinate (HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMCP),hydroxypropyl methylcellulose (HPMC), cellulose acetate phthalate (CAP),cellulose acetate trimellitate (CAT), hydroxypropyl methyl celluloseacetate phthalate, hydroxypropyl methyl cellulose acetate trimellitate,cellulose acetate succinate, methylcellulose acetate succinate,carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methyl cellulose,hydroxypropyl methyl cellulose acetate, hydroxyethylcellulose andothers); dextran; cyclodextrins; homo- and copolymers of vinyllactam,and mixtures thereof; gelatins; hypromellose phthalate; sugars;polyhydric alcohols; polyethylene glycol (PEG); polyethylene oxides;polyoxyethylene derivatives; polyvinyl alcohol; propylene glycolderivatives and the like; SLS; Tween; EUDRAGIT (a methacrylic acid andmethyl methacrylate copolymer); and combinations thereof. The polymermay be water soluble or water insoluble. In certain embodiments, theratio of the sulfasalazine to polymer in the composition is about 5:95wt/wt to 50:50 wt/wt.

In certain embodiments, pharmaceutical compositions comprising amorphousor essentially amorphous sulfasalazine and a pharmaceutically acceptablepolymer are provided. In certain of those embodiments, thepharmaceutical compositions are in the form of a solid dispersion. Incertain embodiments, the pharmaceutically acceptable polymer may beselected from polyvinylpyrrolidone (PVP, including PVP VA64, homo- andcopolymers of polyvinylpyrrolidone and homopolymers or copolymers ofN-vinylpyrrolidone); crospovidone; polyoxyethylene-polyoxypropylenecopolymers (also known as poloxamers); cellulose derivatives (includinghydroxypropyl methyl cellulose acetate succinate (HPMCAS), hydroxypropylmethyl cellulose phthalate (HPMCP), hydroxypropyl methylcellulose(HPMC), cellulose acetate phthalate (CAP), cellulose acetatetrimellitate (CAT), hydroxypropyl methyl cellulose acetate phthalate,hydroxypropyl methyl cellulose acetate trimellitate, cellulose acetatesuccinate, methylcellulose acetate succinate, carboxymethyl ethylcellulose (CMEC), hydroxypropyl methyl cellulose, hydroxypropyl methylcellulose acetate, hydroxyethylcellulose and others); dextran;cyclodextrins; homo- and copolymers of vinyllactam, and mixturesthereof; gelatins; hypromellose phthalate; sugars; polyhydric alcohols;polyethylene glycol (PEG); polyethylene oxides; polyoxyethylenederivatives; polyvinyl alcohol; propylene glycol derivatives and thelike; SLS; Tween; EUDRAGIT (a methacrylic acid and methyl methacrylatecopolymer); and combinations thereof. The polymer may be water solubleor water insoluble. In certain embodiments, the ratio of thesulfasalazine to polymer in the composition is about 5:95 wt/wt to 50:50wt/wt.

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising sulfasalazine and PVP VA64, wherein the ratio ofthe sulfasalazine to PVP VA64 in the composition is about 20:80 wt/wt to30:70 wt/wt. In certain of those embodiments, the ratio of sulfasalazineto PVP VA64 is about 25:75 wt/wt. In certain of those embodiments, thepharmaceutical composition further comprises a pharmaceuticallyacceptable excipient. In certain embodiments, the present inventionprovides pharmaceutical compositions comprising sulfasalazine and PVPVA64, wherein the ratio of the sulfasalazine to PVP VA64 in thecomposition is about 20:80 wt/wt to 30:70 wt/wt; and wherein thesulfasalazine dispersed in the polymer is in an amorphous form. Incertain of those embodiments, the ratio of sulfasalazine to PVP VA64 isabout 25:75 wt/wt. In certain of those embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient.In certain embodiments, the present invention provides pharmaceuticalcompositions comprising sulfasalazine and PVP VA64, wherein the ratio ofthe sulfasalazine to PVP VA64 in the composition is about 40:60 wt/wt toabout 60:40 wt/wt; and wherein the sulfasalazine dispersed in thepolymer is in an amorphous form. In certain of those embodiments, theratio of sulfasalazine to PVP VA64 is about 50:50 wt/wt. In certainembodiments of the methods in this paragraph, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is at least 500 μg/ml, at least 1200 μg/ml or at least2300 μg/ml at a pH of 5.5 determined as in Example 9. In certainembodiments of the methods of this paragraph, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is about 500 μg/ml, about 1200 μg/ml or about 2300 μg/mlat a pH of 5.5 determined as in Example 9. In certain embodiments of themethods of this paragraph, the pharmaceutical composition is formulatedsuch that the in vitro solubility of the sulfasalazine is between about500 μg/ml and about 11,500 μg/ml, inclusive, at a pH of 5.5 determinedas in Example 9. In other embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 7,500 μg/ml, inclusive, at a pH of 5.5determined as in Example 9. In certain embodiments of the methods ofthis paragraph, the pharmaceutical composition is formulated such thatthe in vitro solubility of the sulfasalazine is between about 500 μg/mland about 2500 μg/ml, inclusive, at a pH of 5.5 determined as in Example9. In certain embodiments of the methods of this paragraph, thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is between about 2300 μg/ml and about11,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Inanother embodiment, the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 2300μg/ml and about 5,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9. In other embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 2300 μg/ml and about 7,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9.

In other embodiments, there is provided a spray dried dispersioncomposition comprising sulfasalazine and PVP VA64 polymer, wherein theratio of the sulfasalazine to PVP VA64 in the composition is about 20:80wt/wt to 30:70 wt/wt. In one aspect of the above embodiment, wherein theratio of sulfasalazine to PVP VA64 is about 25:75 wt/wt. In certain ofthe embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable excipient. In other embodiments, there isprovided a spray dried dispersion composition comprising sulfasalazineand PVP VA64 polymer, wherein the ratio of the sulfasalazine to PVP VA64in the composition is about 20:80 wt/wt to 30:70 wt/wt; wherein thesulfasalazine dispersed in the polymer is in an essentially amorphousform. In certain of those embodiments, wherein the sulfasalazinedispersed in the polymer is in an amorphous form. In one aspect of theabove embodiment, wherein the ratio of sulfasalazine to PVP VA64 isabout 25:75 wt/wt. In certain of the embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient.In another aspect of the above, the spray dried dispersion compositionis formulated such that the in vitro solubility of the sulfasalazine isat least 500 μg/ml, at least 1200 μg/ml or at least 2300 μg/ml at a pHof 5.5 determined as in Example 9. In another aspect of the above, thespray dried dispersion composition is formulated such that the in vitrosolubility of the sulfasalazine is about 500 μg/ml, about 1200 μg/ml orabout 2300 μg/ml at a pH of 5.5 determined as in Example 9. In anotheraspect of the above, the spray dried dispersion composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 11,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In another embodiment, the spray drieddispersion composition is formulated such that the in vitro solubilityof the sulfasalazine is between about 500 μg/ml and about 7,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In otherembodiments, the spray dried dispersion composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 500μg/ml and about 5,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9. In other aspects of the above, the spray dried dispersioncomposition is formulated such that the in vitro solubility of thesulfasalazine is between about 500 μg/ml and about 2500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In other aspectsof the above, the spray dried dispersion composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 2300μg/ml and about 11,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9.

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising sulfasalazine and HPMCAS, wherein the ratio ofthe sulfasalazine to HPMCAS in the composition is about 20:80 wt/wt to30:70 wt/wt. In certain of those embodiments, the ratio of sulfasalazineto HPMCAS is about 25:75 wt/wt. In certain embodiments, the presentinvention provides pharmaceutical compositions comprising sulfasalazineand HPMCAS, wherein the ratio of the sulfasalazine to HPMCAS in thecomposition is about 20:80 wt/wt to 30:70 wt/wt; wherein thesulfasalazine dispersed in the polymer is in an amorphous form. Incertain of those embodiments, the ratio of sulfasalazine to HPMCAS isabout 25:75 wt/wt. In certain embodiments of the pharmaceuticalcompositions of this paragraph, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine is atleast 500 μg/ml, at least 1200 μg/ml or at least 2300 μg/ml at a pH of5.5 determined as in Example 9. In certain embodiments of thepharmaceutical compositions of this paragraph, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is about 500 μg/ml, about 1200 μg/ml or about 2300 μg/mlat a pH of 5.5 determined as in Example 9. In certain embodiments of thepharmaceutical compositions of this paragraph, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is between about 500 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In other aspectsof the above, the pharmaceutical composition is formulated such that thein vitro solubility of the sulfasalazine is between about 500 μg/ml andabout 2500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9.In another aspect, the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 2300μg/ml and about 5,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9. In another aspect, pharmaceutical composition is formulatedsuch that the in vitro solubility of the sulfasalazine is between about2300 μg/ml and about 7,500 μg/ml, inclusive, at a pH of 5.5 determinedas in Example 9. In other aspects of the above, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9.

In certain embodiments, the present invention provides spray drieddispersion compositions comprising sulfasalazine and HPMCAS, wherein theratio of the sulfasalazine to HPMCAS in the composition is about 20:80wt/wt to 30:70 wt/wt. In certain of those embodiments, the ratio ofsulfasalazine to HPMCAS is about 25:75 wt/wt. In certain embodiments,the present invention provides spray dried dispersion compositionscomprising sulfasalazine and HPMCAS, wherein the ratio of thesulfasalazine to HPMCAS in the composition is about 20:80 wt/wt to 30:70wt/wt; wherein the sulfasalazine dispersed in the polymer is in anessentially amorphous form. In certain of those embodiments, thesulfasalazine dispersed in the polymer is in an amorphous form. In someembodiments, there is provided a spray dried dispersion compositioncomprising sulfasalazine and HPMCAS polymer, wherein the ratio of thesulfasalazine to HPMCAS in the composition is about 20:80 wt/wt to 30:70wt/wt; wherein the sulfasalazine dispersed in the polymer is in anessentially amorphous form and the spray dried dispersion composition isformulated such that the in vitro solubility of the sulfasalazine is atleast 500 μg/ml, at least 1200 μg/ml or at least 2300 μg/ml at a pH of5.5 determined as in Example 9. In one aspect of the above embodiment,the ratio of sulfasalazine to HPMCAS is about 25:75 wt/wt. In someembodiments, there is provided a spray dried dispersion compositioncomprising sulfasalazine and HPMCAS, wherein the ratio of thesulfasalazine to HPMCAS in the composition is about 20:80 wt/wt to 30:70wt/wt; wherein the sulfasalazine dispersed in the polymer is in anessentially amorphous form and the spray dried dispersion composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 11,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In another aspect, the spray drieddispersion composition is formulated such that the in vitro solubilityof the sulfasalazine is between about 500 μg/ml and about 7,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In certain ofthose embodiments, the spray dried dispersion composition is formulatedsuch that the in vitro solubility of the sulfasalazine is between about500 μg/ml and about 2500 μg/ml, inclusive, at a pH of 5.5 determined asin Example 9. In other of those embodiments, the spray dried dispersioncomposition is formulated such that the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In certainembodiments of the above embodiments, the ratio of sulfasalazine toHPMCAS is about 25:75 wt/wt.

In another embodiment, there is provided a spray dried dispersioncomposition comprising sulfasalazine and a pharmaceutically acceptablepolymer. In certain embodiments, the pharmaceutically acceptable polymermay be selected from polyvinylpyrrolidone (PVP, including PVP VA64,homo- and copolymers of polyvinylpyrrolidone and homopolymers orcopolymers of N-vinylpyrrolidone); crospovidone;polyoxyethylene-polyoxypropylene copolymers (also known as poloxamers);cellulose derivatives (including hydroxypropyl methyl cellulose acetatesuccinate (HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMCP),hydroxypropyl methylcellulose (HPMC), cellulose acetate phthalate (CAP),cellulose acetate trimellitate (CAT), hydroxypropyl methyl celluloseacetate phthalate, hydroxypropyl methyl cellulose acetate trimellitate,cellulose acetate succinate, methylcellulose acetate succinate,carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methyl cellulose,hydroxypropyl methyl cellulose acetate, hydroxyethylcellulose andothers); dextran; cyclodextrins; homo- and copolymers of vinyllactam,and mixtures thereof; gelatins; hypromellose phthalate; sugars;polyhydric alcohols; polyethylene glycol (PEG); polyethylene oxides;polyoxyethylene derivatives; polyvinyl alcohol; propylene glycolderivatives and the like; SLS; Tween; EUDRAGIT (a methacrylic acid andmethyl methacrylate copolymer); and combinations thereof. The polymermay be water soluble or water insoluble. In certain embodiments, theratio of the sulfasalazine to polymer in the composition is about 5:95wt/wt to 50:50 wt/wt. In certain of those embodiments, the sulfasalazinedispersed in the polymer is in an essentially amorphous form.

In certain embodiments of the compositions and methods provided in thisapplication, the sulfasalazine is in an amorphous form or an essentiallyamorphous form. In certain embodiments of the compositions and methodsprovided in this application, the composition comprises PVP VA64 orHPMCAS. In certain of those embodiments, the ratio of the sulfasalazineto PVP VA64 or HPMCAS in the composition is about 20:80 wt/wt to 30:70wt/wt or is about 25:75 wt/wt. In certain other of those embodiments,the ratio of the sulfasalazine to PVP VA64 in the composition is about40:60 wt/wt to about 60:40 wt/wt or is about 50:50 wt/wt.

Treating Neurodegenerative Diseases and Disorders with the Formulationsof the Invention

In certain embodiments, the formulations of the present invention can beused in the treatment of neurodegenerative diseases and disorders aswell as certain other diseases and disorders. For example, the variousformulations and compositions comprising sulfasalazine described in thisapplication can be used in the treatment of P-MS, ALS, neuropathic painand other neurodegenerative diseases and disorders.

In certain embodiments, the application provides methods for treatingP-MS in a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is at least 500 μg/ml, at least 1200μg/ml or at least 2300 μg/ml at a pH of 5.5 determined as in Example 9.In certain embodiments, the application provides methods for treatingP-MS in a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is at least 2300 μg/ml at a pH of 5.5 asdetermined in Example 9. In certain embodiments, the applicationprovides methods for treating P-MS in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isabout 500 μg/ml, about 1200 μg/ml or about 2300 μg/ml at a pH of 5.5 asdetermined in Example 9. In certain embodiments, the applicationprovides methods for treating P-MS in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 11,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In certain of those embodiments, thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about2500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Inanother embodiment, the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 2300μg/ml and about 5,500 μg/ml, inclusive, at a pH of 5.5 determined as inExample 9. In other embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 2300 μg/ml and about 7,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In certain other of those embodiments,the pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is between about 2300 μg/ml and about11,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Incertain embodiments described in this paragraph, the pharmaceuticalcomposition is one of the pharmaceutical compositions provided by thepresent application. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform.

In certain embodiments, methods are provided for treating P-MS in apatient comprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is at least 2 times higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain of thoseembodiments, the pharmaceutical composition is formulated such that thein vitro solubility of the sulfasalazine at a pH of 5.5 as determined inExample 9 is at least 5 times or at least 8.8 times higher than the invitro solubility of crystalline sulfasalazine at a pH of 5.5 by AUCanalysis. In certain embodiments, methods are provided for treating P-MSin a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is about 2 times, about 5 times, or about 8.8 times higher than the invitro solubility of crystalline sulfasalazine at a pH of 5.5 by AUCanalysis. In certain embodiments, methods are provided for treating P-MSin a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is between about 2 times and about 44 times, inclusive, higher thanthe in vitro solubility of crystalline sulfasalazine at a pH of 5.5 byAUC analysis. In certain of those embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is betweenabout 2 times and about 8.8 times, inclusive, higher than the in vitrosolubility of crystalline sulfasalazine at a pH of 5.5 by AUC analysis.In certain other embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined as in Example 9 is between about 8.8 times andabout 44 times, inclusive, higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5 by AUC analysis. In certainembodiments described in this paragraph, the pharmaceutical compositionis one of the pharmaceutical compositions provided by the presentapplication. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform.

In some embodiments, there are provided methods for treating a patientwith P-MS comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe sulfasalazine is in an essentially amorphous form. In one aspect ofthe method, the in vitro solubility of the sulfasalazine from thecomposition is at least 500 μg/ml at a pH of 5.5 when measured by themethod of Example 9. In another aspect of the invention, the in vitrosolubility of the sulfasalazine is at least 1200 μg/ml or at least 2300μg/ml. In certain embodiments, the application provides methods fortreating P-MS in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of essentially amorphous sulfasalazine and apharmaceutically acceptable excipient, wherein the in vitro solubilityof the sulfasalazine is about 500 μg/ml, about 1200 μg/ml or about 2300μg/ml at a pH of 5.5 as determined in Example 9. In other aspects of theinvention, the in vitro solubility of the sulfasalazine is between about500 μg/ml and about 11,500 μg/ml, inclusive. In other aspects of theinvention, the in vitro solubility of the sulfasalazine is between about500 μg/ml and about 2500 μg/ml, inclusive. In another aspect of theabove, the in vitro solubility of the sulfasalazine is between about2300 μg/ml and about 5,500 μg/ml, inclusive. In another aspect of theabove, the in vitro solubility of the sulfasalazine is between about2300 μg/ml and about 7,500 μg/ml, inclusive. In other aspects of theinvention, the in vitro solubility of the sulfasalazine is between about2300 μg/ml and about 11,500 μg/ml, inclusive.

In another aspect of the method, the pharmaceutical compositioncomprises a pharmaceutically acceptable polymer. In certain of thoseembodiments, the pharmaceutically acceptable polymeris a pyrrolidonepolymer. In another aspect of the method, the pyrrolidone polymer isselected from polyvinylpyrrolidone, vinylpyrrolidone-vinyl acetate andcrospovidone. In yet another aspect of the method, the pyrrolidonepolymer is PVP VA64. In another aspect of the above method, the ratio ofthe sulfasalazine to PVP VA64 in the pharmaceutical composition is about20:80 wt/wt to 30:70 wt/wt. In another aspect, the ratio ofsulfasalazine to PVP VA64 is about 25:75 wt/wt. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In certain embodiments, the application provides methods for treatingALS in a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is at least 500 μg/ml, at least 1200μg/ml or at least 2300 μg/ml at a pH of 5.5 determined as in Example 9.In certain embodiments, the application provides methods for treatingALS in a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is at least 2300 μg/ml at a pH of 5.5 asdetermined in Example 9. In certain embodiments, the applicationprovides methods for treating ALS in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isabout 500 μg/ml, about 1200 μg/ml or about 2300 μg/ml at a pH of 5.5 asdetermined in Example 9. In certain embodiments, the applicationprovides methods for treating ALS in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 11,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In certain of those embodiments, thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about2500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Incertain of those embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 2300 μg/ml and about 11,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In another embodiment of the above, thein vitro solubility of the sulfasalazine is between about 2300 μg/ml andabout 7,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9.In another embodiment of the above, the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 5,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In certainembodiments described in this paragraph, the pharmaceutical compositionis one of the pharmaceutical compositions provided by the presentapplication. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform.

In certain embodiments, methods are provided for treating ALS in apatient comprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is at least 2 times higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain of thoseembodiments, the pharmaceutical composition is formulated such that thein vitro solubility of the sulfasalazine at a pH of 5.5 as determined inExample 9 is at least 5 times or at least 8.8 times higher than the invitro solubility of crystalline sulfasalazine at a pH of 5.5 by AUCanalysis. In certain embodiments, methods are provided for treating ALSin a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is about 2 times, about 5 times, or about 8.8 times higher than the invitro solubility of crystalline sulfasalazine at a pH of 5.5 by AUCanalysis. In certain embodiments, methods are provided for treating ALSin a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is between about 2 times and about 44 times, inclusive, higher thanthe in vitro solubility of crystalline sulfasalazine at a pH of 5.5 byAUC analysis. In certain of those embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is betweenabout 2 times and about 8.8 times, inclusive, higher than the in vitrosolubility of crystalline sulfasalazine at a pH of 5.5 by AUC analysis.In certain other embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined as in Example 9 is between about 8.8 times andabout 44 times, inclusive, higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5 by AUC analysis. In certainembodiments described in this paragraph, the pharmaceutical compositionis one of the pharmaceutical compositions provided by the presentapplication. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform.

In some embodiments, there are provided methods for treating a patientwith ALS comprising orally administering to the patient with ALS apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe sulfasalazine is in an essentially amorphous form. In certainaspects of the method, the in vitro solubility of the sulfasalazine fromthe composition is at least 500 μg/ml at a pH of 5.5 when measured bythe method of Example 9. In certain of those aspects, the in vitrosolubility of the sulfasalazine is at least 1200 μg/ml or at least 2300μg/ml. In certain embodiments, the application provides methods fortreating ALS in a patient comprising orally administering to the patienta pharmaceutical composition comprising a therapeutically effectiveamount of essentially amorphous sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the in vitro solubility of thesulfasalazine is about 500 μg/ml, about 1200 μg/ml or about 2300 μg/mlat a pH of 5.5 as determined in Example 9. In some embodiments, thereare provided methods for treating a patient with ALS comprising orallyadministering to the patient with ALS a pharmaceutical compositioncomprising a therapeutically effective amount of essentially amorphoussulfasalazine and a pharmaceutically acceptable excipient, wherein thein vitro solubility of the sulfasalazine is between about 500 μg/ml andabout 11,500 μg/ml, inclusive. In another embodiment, the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about7,500 μg/ml, inclusive. In another embodiment of the above, the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about5,500 μg/ml, inclusive. In certain of those embodiments, the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about2500 μg/ml, inclusive. In other of those embodiments, the in vitrosolubility of the sulfasalazine is between about 2300 μg/ml and about11,500 μg/ml, inclusive. In another aspect of the method, thepharmaceutical composition comprises a pharmaceutically acceptablepolymer. In certain of those embodiments, the pharmaceuticallyacceptable polymer comprises a pyrrolidone polymer. In another aspect ofthe method, the pyrrolidone polymer is selected frompolyvinylpyrrolidone, vinylpyrrolidone-vinyl acetate and crospovidone.In yet another aspect of the method, the pyrrolidone polymer is PVPVA64. In another aspect of the above method, the ratio of thesulfasalazine to PVP VA64 in the pharmaceutical composition is about20:80 wt/wt to 30:70 wt/wt. In another aspect, the ratio ofsulfasalazine to PVP VA64 is about 25:75 wt/wt. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In certain embodiments, the application provides methods for treatingneuropathic pain in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine is at least 500 μg/ml,at least 1200 μg/ml or at least 2300 μg/ml at a pH of 5.5 determined asin Example 9. In certain embodiments, the application provides methodsfor treating neuropathic pain in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isabout 500 μg/ml, about 1200 μg/ml or about 2300 μg/ml at a pH of 5.5 asdetermined in Example 9. In certain embodiments, the applicationprovides methods for treating neuropathic pain in a patient comprisingorally administering to the patient a pharmaceutical compositioncomprising a therapeutically effective amount of sulfasalazine and apharmaceutically acceptable excipient, wherein the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is between about 500 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 determined as in Example 9. In anotherembodiment, the in vitro solubility of the sulfasalazine is betweenabout 500 μg/ml and about 7,500 μg/ml, inclusive, at a pH of 5.5determined as in Example 9. In another embodiment, the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about5,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Incertain of those embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 2500 μg/ml, inclusive, at a pH of 5.5determined as in Example 9. In certain of those embodiments, thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is between about 2300 μg/ml and about11,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Insome of the embodiments in this paragraph, the neuropathic pain resultsfrom painful diabetic neuropathy. In certain embodiments, theneuropathic pain manifests as dysesthesia. In certain embodiments, theneuropathic pain manifests as allodynia. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In certain embodiments, methods are provided for treating neuropathicpain in a patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined in Example9 is at least 2 times higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain of thoseembodiments, the pharmaceutical composition is formulated such that thein vitro solubility of the sulfasalazine at a pH of 5.5 as determined inExample 9 is at least 5 times or at least 8.8 times higher than the invitro solubility of crystalline sulfasalazine at a pH of 5.5 by AUCanalysis. In certain embodiments, methods are provided for treatingneuropathic pain in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine at a pH of 5.5 asdetermined in Example 9 is about 2 times, about 5 times, or about 8.8times higher than the in vitro solubility of crystalline sulfasalazineat a pH of 5.5 by AUC analysis. In certain embodiments, methods areprovided for treating neuropathic pain in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined in Example 9 is between about 2 times and about44 times, inclusive, higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain of thoseembodiments, the pharmaceutical composition is formulated such that thein vitro solubility of the sulfasalazine at a pH of 5.5 as determined asin Example 9 is between about 2 times and about 8.8 times, inclusive,higher than the in vitro solubility of crystalline sulfasalazine at a pHof 5.5 by AUC analysis. In certain other embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is betweenabout 8.8 times and about 44 times, inclusive, higher than the in vitrosolubility of crystalline sulfasalazine at a pH of 5.5 by AUC analysis.In some of the embodiments in this paragraph, the neuropathic painresults from painful diabetic neuropathy. In certain embodiments, theneuropathic pain manifests as dysesthesia. In certain embodiments, theneuropathic pain manifests as allodynia. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In some embodiments, there are provided methods for treating a patientwith neuropathic pain comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe sulfasalazine is in an essentially amorphous form. In certainaspects of the method, the in vitro solubility of the sulfasalazine fromthe composition is at least 500 μg/ml at a pH of 5.5 when measured bythe method of Example 9. In certain of those aspects, the in vitrosolubility of the sulfasalazine is at least 1200 μg/ml or at least 2300μg/ml. In certain embodiments, the application provides methods fortreating neuropathic pain in a patient comprising orally administeringto the patient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the in vitro solubility of the sulfasalazine is about500 μg/ml, about 1200 μg/ml or about 2300 μg/ml at a pH of 5.5 asdetermined in Example 9. In some embodiments, there are provided methodsfor treating a patient with neuropathic pain comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of essentially amorphous sulfasalazineand a pharmaceutically acceptable excipient, wherein the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about11,500 μg/ml, inclusive. In certain of those embodiments, the in vitrosolubility of the sulfasalazine is between about 500 μg/ml and about2500 μg/ml, inclusive. In another embodiment, the in vitro solubility ofthe sulfasalazine is between about 2300 μg/ml and about 7,500 μg/ml,inclusive. In other of those embodiments, the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 11,500 μg/ml,inclusive. In another aspect of the method, the pharmaceuticalcomposition comprises a pharmaceutically acceptable polymer. In certainof those embodiments, the pharmaceutically acceptable polymer is apyrrolidone polymer. In another aspect of the method, the pyrrolidonepolymer is selected from polyvinylpyrrolidone, vinylpyrrolidone-vinylacetate and crospovidone. In yet another aspect of the method, thepyrrolidone polymer is PVP VA64. In another aspect of the above method,the ratio of the sulfasalazine to PVP VA64 in the pharmaceuticalcomposition is about 20:80 wt/wt to 30:70 wt/wt and wherein thesulfasalazine dispersed in the polymer is in an amorphous form. Inanother aspect, the ratio of sulfasalazine to PVP VA64 is about 25:75wt/wt. In some of the embodiments in this paragraph, the neuropathicpain results from painful diabetic neuropathy. In certain embodiments,the neuropathic pain manifests as dysesthesia. In certain embodiments,the neuropathic pain manifests as allodynia. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In other embodiments, there are provided methods for treating aneurodegenerative disease or disorder in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine is atleast 500 μg/ml, at least 1200 μg/ml, or at least 2300 μg/ml at a pH of5.5 as determined as in Example 9. In other embodiments, there areprovided methods for treating a neurodegenerative disease or disorder ina patient comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and a pharmaceutically acceptable excipient, whereinthe pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is about 500 μg/ml, about 1200 μg/ml, orabout 2300 μg/ml at a pH of 5.5 as determined as in Example 9. In otherembodiments, there are provided methods for treating a neurodegenerativedisease or disorder in a patient comprising orally administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 500μg/ml and about 11,500 μg/ml, inclusive, at a pH of 5.5 as determined asin Example 9. In another embodiment, the in vitro solubility of thesulfasalazine is between about 500 μg/ml and about 7,500 μg/ml,inclusive, at a pH of 5.5 as determined as in Example 9. In certain ofthose embodiments, the pharmaceutical composition is formulated suchthat the in vitro solubility of the sulfasalazine is between about 500μg/ml and about 2500 μg/ml, inclusive, at a pH of 5.5 as determined asin Example 9. In other of those embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine is between about 2300 μg/ml and about 11,500 μg/ml,inclusive, at a pH of 5.5 as determined as in Example 9. In certainembodiments described in this paragraph, the pharmaceutical compositionis one of the pharmaceutical compositions provided by the presentapplication. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform. In certain embodiments in this paragraph, the neurodegenerativedisease is selected from Parkinson's disease, Alzheimer's disease,epilepsy, traumatic brain injury, Huntington's disease, ischemic stroke,Rett Syndrome, Frontotemporal Dementia, HIV-associated Dementia,Tuberous Sclerosis and Alexander disease.

In other embodiments, there are provided methods for treating aneurodegenerative disease or disorder in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined as in Example 9 is at least 2 times or 5 timeshigher than the in vitro solubility of crystalline sulfasalazine at a pHof 5.5. In certain of those embodiments, the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined as in Example 9 is at least8.8 times higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5. In other embodiments, there are providedmethods for treating a neurodegenerative disease or disorder in apatient comprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined as inExample 9 is between about 2 times and about 44 times, inclusive, higherthan the in vitro solubility of crystalline sulfasalazine at a pH of5.5. In certain of those embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined as in Example 9 is between about 2 times andabout 8.8 times, inclusive, higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5. In certain other embodiments,the pharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine at a pH of 5.5 as determined as inExample 9 is between about 8.8 times and about 44 times, inclusive,higher than the in vitro solubility of crystalline sulfasalazine at a pHof 5.5. In certain embodiments described in this paragraph, thepharmaceutical composition is one of the pharmaceutical compositionsprovided by the present application. In certain of those embodiments,the pharmaceutical composition is in an oral dosage form or in a spraydried dispersion form. In certain embodiments in this paragraph, theneurodegenerative disease is selected from Parkinson's disease,Alzheimer's disease, epilepsy, traumatic brain injury, Huntington'sdisease, ischemic stroke, Rett Syndrome, Frontotemporal Dementia,HIV-associated Dementia, Tuberous Sclerosis and Alexander disease.

In some embodiments, the present invention provides methods for treatinga patient with a neurodegenerative disease or disorder in a patientcomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutical acceptable excipient, wherein thesulfasalazine is in an essentially amorphous form. In one aspect of themethod, the in vitro solubility of the sulfasalazine from thecomposition is at least 500 μg/ml at a pH of 5.5 when measured by themethod of Example 9. In another aspect of the invention, the in vitrosolubility of the sulfasalazine from the composition is at least 1200μg/ml or at least 2300 μg/ml. In other aspects of the invention, the invitro solubility of the sulfasalazine from the composition is betweenabout 500 μg/ml and about 11,500 μg/ml, inclusive. In another aspect,the in vitro solubility of the sulfasalazine from the composition isbetween about 500 μg/ml and about 7,500 μg/ml, inclusive. In otheraspects of the invention, the in vitro solubility of the sulfasalazinefrom the composition is between about 500 μg/ml and about 2500 μg/ml. Inother aspects of the invention, the in vitro solubility of thesulfasalazine from the composition is between about 2300 μg/ml and about11,500 μg/ml. In another aspect of the method, the pharmaceuticalcomposition comprises a pharmaceutically acceptable polymer. In certainof those embodiments, the pharmaceutically acceptable polymer is apyrrolidone polymer. In another aspect of the method, the pyrrolidonepolymer is selected from polyvinylpyrrolidone, vinylpyrrolidone-vinylacetate and crospovidone. In yet another aspect of the method, thepyrrolidone polymer is PVP VA64. In another aspect of the above method,the ratio of the sulfasalazine to PVP VA64 in the pharmaceuticalcomposition is about 20:80 wt/wt to 30:70 wt/wt. In another aspect, theratio of sulfasalazine to PVP VA64 is about 25:75 wt/wt. In certainembodiments in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form. In certainembodiments in this paragraph, the neurodegenerative disease is selectedfrom Parkinson's disease, Alzheimer's disease, epilepsy, traumatic braininjury, Huntington's disease, ischemic stroke, Rett Syndrome,Frontotemporal Dementia, HIV-associated Dementia, Tuberous Sclerosis andAlexander disease.

In other embodiments, there are provided methods for treating a patientwith P-MS comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and PVP VA64; wherein the ratio of sulfasalazine to PVPVA64 is about 20:80 wt/wt to 30:70 wt/wt. In certain of thoseembodiments, the sulfasalazine dispersed in the polymer is in anessentially amorphous form. In certain of those embodiments, thesulfasalazine is dispersed in the polymer is in an amorphous form. Incertain of the embodiments in this paragraph, the ratio of sulfasalazineto PVP VA64 is about 25:75 wt/wt. In certain embodiments in thisparagraph, the pharmaceutical composition further comprises apharmaceutically acceptable excipient.

In certain embodiments, methods are provided for treating a patient withP-MS comprising orally administering to the patient a pharmaceuticalcomposition comprising sulfasalazine and HPMCAS, wherein the ratio ofthe sulfasalazine to HPMCAS in the composition is about 20:80 wt/wt to30:70 wt/wt and wherein the sulfasalazine dispersed in the polymer is inan essentially amorphous form. In certain of those embodiments, thesulfasalazine is dispersed in the polymer is in an amorphous form. Incertain embodiments, the ratio of sulfasalazine to HPMCAS is about 25:75wt/wt. In certain embodiments in this paragraph, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient.

In other embodiments, there are provided methods for treating a patientwith ALS comprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and PVP VA64; wherein the ratio of sulfasalazine to PVPVA64 is about 20:80 wt/wt to 30:70 wt/wt and wherein the sulfasalazinedispersed in the polymer is in an essentially amorphous form. In certainof those embodiments, the sulfasalazine is dispersed in the polymer isin an amorphous form. In certain embodiments, the ratio of sulfasalazineto PVP VA64 is about 25:75 wt/wt. In certain embodiments in thisparagraph, the pharmaceutical composition further comprises apharmaceutically acceptable excipient.

In certain embodiments, methods are provided for treating a patient withALS comprising orally administering to the patient a pharmaceuticalcomposition comprising sulfasalazine and HPMCAS; wherein the ratio ofthe sulfasalazine to HPMCAS in the composition is about 20:80 wt/wt to30:70 wt/wt and wherein the sulfasalazine dispersed in the polymer is inan essentially amorphous form. In certain of those embodiments, thesulfasalazine is dispersed in the polymer is in an amorphous form. Incertain embodiments, the ratio of sulfasalazine to HPMCAS is about 25:75wt/wt. In certain embodiments in this paragraph, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient.

In other embodiments, there are provided methods for treating a patientwith neuropathic pain comprising orally administering to the patient apharmaceutical composition comprising a therapeutically effective amountof sulfasalazine and PVP VA64; wherein the ratio of sulfasalazine to PVPVA64 is about 20:80 wt/wt to 30:70 wt/wt; wherein the sulfasalazinedispersed in the polymer is in an essentially amorphous form. In certainof those embodiments, the sulfasalazine is dispersed in the polymer isin an amorphous form. In certain embodiments, the ratio of sulfasalazineto PVP VA64 is about 25:75 wt/wt. In some of the embodiments in thisparagraph, the neuropathic pain is diabetic neuropathic pain. In certainembodiments, the neuropathic pain manifests as dysesthesia. In certainembodiments, the neuropathic pain manifests as allodynia. In certainembodiments in this paragraph, the pharmaceutical composition furthercomprises a pharmaceutically acceptable excipient.

In certain embodiments, methods are provided for treating a patient withneuropathic pain comprising orally administering to the patient apharmaceutical composition comprising sulfasalazine and HPMCAS; whereinthe ratio of the sulfasalazine to HPMCAS in the composition is about20:80 wt/wt to 30:70 wt/wt and wherein the sulfasalazine dispersed inthe polymer is in an essentially amorphous form. In certain of thoseembodiments, the sulfasalazine is dispersed in the polymer is in anamorphous form. In certain embodiments, the ratio of sulfasalazine toHPMCAS is about 25:75 wt/wt. In some of the embodiments in thisparagraph, the neuropathic pain results from painful diabeticneuropathy. In certain embodiments, the neuropathic pain manifests asdysesthesia. In certain embodiments, the neuropathic pain manifests asallodynia. In certain embodiments in this paragraph, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient.

In other embodiments, there are provided methods for treating aneurodegenerative disease or disorder in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and PVP VA64; whereinthe ratio of sulfasalazine to PVP VA64 is about 20:80 wt/wt to 30:70wt/wt and wherein the sulfasalazine dispersed in the polymer is in anessentially amorphous form. In certain of those embodiments, thesulfasalazine is dispersed in the polymer is in an amorphous form. Incertain of those embodiments, the ratio of sulfasalazine to PVP VA64 isabout 25:75 wt/wt. In certain embodiments in this paragraph, thepharmaceutical composition further comprises a pharmaceuticallyacceptable excipient. In certain embodiments in this paragraph, theneurodegenerative disease is selected from Parkinson's disease,Alzheimer's disease, epilepsy, traumatic brain injury, Huntington'sdisease, ischemic stroke, Rett Syndrome, Frontotemporal Dementia,HIV-associated Dementia, Tuberous Sclerosis and Alexander disease.

In other embodiments, there are provided methods for treating aneurodegenerative disease or disorder in a patient comprising orallyadministering to the patient a pharmaceutical compositions comprisingsulfasalazine and HPMCAS wherein the ratio of the sulfasalazine toHPMCAS in the composition is about 20:80 wt/wt to 30:70 wt/wt. Incertain of those embodiments, the sulfasalazine is dispersed in anessentially amorphous form. In certain of those embodiments, thesulfasalazine is dispersed in the polymer is in an amorphous form. Incertain of those embodiments, the ratio of sulfasalazine to HPMCAS isabout 25:75 wt/wt. In certain embodiments in this paragraph, thepharmaceutical composition further comprises a pharmaceuticallyacceptable excipient. In certain embodiments in this paragraph, theneurodegenerative disease is selected from Parkinson's disease,Alzheimer's disease, epilepsy, traumatic brain injury, Huntington'sdisease, ischemic stroke, Rett Syndrome, Frontotemporal Dementia,HIV-associated Dementia, Tuberous Sclerosis and Alexander disease.

In certain embodiments, methods are provided for lowering excessivelevels of glutamate in a patient with a neurodegenerative diseasecomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pyrrolidone polymer, wherein the ratio of thesulfasalazine to the pyrrolidone polymer in the composition is about20:80 wt/wt to 30:70 wt/wt and wherein the sulfasalazine dispersed inthe polymer is in an essentially amorphous form. In certain of thoseembodiments, the sulfasalazine is dispersed in the polymer is in anamorphous form. In certain of those embodiments, the ratio ofsulfasalazine to pyrrolidone polymer is about 25:75 wt/wt. In certainembodiments in this paragraph, the pharmaceutical composition furthercomprises a pharmaceutically acceptable excipient.

In one aspect of each of the above embodiments, the neurodegenerativedisease or disorder is selected from the group consisting of epilepsy,stroke or traumatic brain injury. In another aspect, theneurodegenerative disease or disorder is Parkinson's disease (PD),Alzheimer's disease (AD), or Huntington's. In another aspect of each ofthe above embodiments, the neurodegenerative disease or disorder isprogressive MS (P-MS). In another aspect of each of the aboveembodiments, the neurodegenerative disease or disorder is amyotrophiclateral sclerosis (ALS). In another aspect of each of the aboveembodiments, the neurodegenerative disease or disorder is neuropathicpain. In another aspect of each of the above embodiments, thesulfasalazine is amorphous sulfasalazine. In another aspect of each ofthe above embodiments, the in vitro solubility of the sulfasalazine isat least 500 μg/ml or at least 1200 μg/ml at a pH of 5.5 as determinedas in Example 9. In another aspect of each of the above embodiments, themethods comprise the use of one of the above-cited pharmaceuticalcompositions comprising sulfasalazine.

Combination Treatment Methods

In certain aspects of the invention, a patient with ALS is alsoadministered riluzole in addition to a pharmaceutical composition of theinvention. In certain of these embodiments, the riluzole is administeredto the patient concurrently with the pharmaceutical composition. Incertain embodiments, the riluzole is administered to the patient atdifferent times than the pharmaceutical composition.

In certain aspects of the invention, a patient with P-MS is alsoadministered Mitoxantrone, Gilenya, Masitinib, Siponimod, Tcelna,Tecfidera, Lemtrada, Laquinimod, Daclizumab, Ocrelizumab, Cladribine,Daclizumab, Tysabri, Campath, Rituximab, Fingolimod, Azathioprine orIbudilast in addition to a pharmaceutical composition of the invention.In certain of these embodiments, the Mitoxantrone, Gilenya, Masitinib,Siponimod, Tcelna, Tecfidera, Lemtrada, Laquinimod, Daclizumab,Ocrelizumab, Cladribine, Daclizumab, Tysabri, Campath, Rituximab,Fingolimod, Azathioprine or Ibudilast is administered to the patientconcurrently with the pharmaceutical composition. In certainembodiments, the Mitoxantrone, Gilenya, Masitinib, Siponimod, Tcelna,Tecfidera, Lemtrada, Laquinimod, Daclizumab, Ocrelizumab, Cladribine,Daclizumab, Tysabri, Campath, Rituximab, Fingolimod, Azathioprine orIbudilast is administered to the patient at different times than thepharmaceutical composition.

Dosing Regimens for the Treatment of P-MS

In certain embodiments, the present invention provides methods oftreating P-MS in patients by administering a therapeutically effectiveamount of a system x_(c) ⁻ inhibitor to such patients. In certain ofthose embodiments, the system x_(c) ⁻ inhibitor is sulfasalazine.Previous work has tested use of sulfasalazine for treatment of multiplesclerosis (both RR-MS and P-MS) in humans, e.g. Noseworthy et al,Neurology 15: 1342-1352 (1998). In this work, patients were treated with2 grams of sulfasalazine per day, the typical maintenance dose used fornon-CNS diseases, such as rheumatoid arthritis, e.g. Khan et al, Gut21:232-240 (1980). Sulfasalazine did not slow disease progression in theRR-MS sub-group. In the P-MS subgroup, patients treated withsulfasalazine had a statistically significant reduction in theiraccumulation of disability, which the authors attributed to a “realtreatment effect.” See id. at p. 1346. However, no further clinicaltrials of sulfasalazine for the treatment of either P-MS or RR-MS havebeen performed. Other previous work demonstrated that a 2 g oral dose ofsulfasalazine administered to humans produced plasma levels above 10μg/ml that were maintained for only approximately 7 hours in people withthe ABCG2 genotype (421C/C) (see Yamasaki et al, Clin. Pharmac. Therap.84: 95-103 (2007)), which is the predominant ABCG2 genotype in EuropeanCaucasian and African American populations (77%-90%) see, e.g., de Jonget al, Clin. Cancer Res. 10:5889-5894 (2004). Prior work had also shownthat the anti-epileptic effect of sulfasalazine administered to a mousemodel at a dose of approximately 260-320 mg/kg intraperitoneal (“IP”)disappears by three hours after administration (see Buckingham et al,Nat Med. 17:1269-1274 (2011)). As experiments described herein indicatethat the plasma level of sulfasalazine in a mouse administered a 200mg/kg dose IP of sulfasalazine (approximately 30-60% lower dose than theBuckingham study) is about 6 μg/ml three hours after administration (seeExample 4), the inventors determined that a plasma level ofsulfasalazine of at least approximately 8-10 μg/ml (adjusted for dosedifferences) is needed is needed for a therapeutic effect bysulfasalazine on the system x_(c) ⁻ in the CNS compartment. Based onthis, the inventors hypothesize that the P-MS patients treated withsulfasalazine in the Noseworthy study were under-dosed. Thus, theinvention also provides methods of treating P-MS with sulfasalazineusing improved dosing regimens and formulations.

In certain embodiments, the present invention provides methods fortreating P-MS in a patient comprising administering to the patient apharmaceutical composition comprising sulfasalazine, wherein thesulfasalazine is dosed at levels and/or frequencies sufficient toproduce improved therapeutic effects. In certain embodiments, methodsare provided for treating a patient with P-MS comprising orallyadministering to the patient a pharmaceutical composition comprisingsulfasalazine and a pharmaceutically acceptable excipient, wherein thedose of the pharmaceutical composition is sufficient to maintain aplasma level of sulfasalazine in the patient effective for treating P-MSfor at least 14 total hours a day. In certain of those embodiments, aplasma level of sulfasalazine in the patient effective for treating P-MSis maintained for between 21 and 24, inclusive, total hours a day. Incertain of those embodiments, a plasma level of sulfasalazine in thepatient effective for treating P-MS is maintained for 24 hours a day.

In certain embodiments, methods are provided for treating a patient withP-MS comprising orally administering to the patient a pharmaceuticalcomposition comprising sulfasalazine and a pharmaceutically acceptableexcipient, wherein the dose of the pharmaceutical composition issufficient to maintain a plasma level of sulfasalazine of at least 8μg/ml for at least 14 total hours a day. In certain of thoseembodiments, a plasma level of sulfasalazine of at least 8 μg/ml ismaintained for between 21 and 24, inclusive, total hours a day. Incertain of those embodiments, a plasma level of sulfasalazine of atleast 8 μg/ml is maintained for 24 hours a day. In certain embodimentsin this paragraph, the dose of the pharmaceutical composition issufficient to maintain a plasma level of sulfasalazine of between about8 μg/ml and about 30 μg/ml, inclusive, or between about 8 μg/ml andabout 16 μg/ml, inclusive, or between about 10 μg/ml and about 16 μg/ml,inclusive, for the given amount of time.

In certain embodiments, the present invention provides methods fortreating a patient with P-MS comprising administering to the patient apharmaceutical composition comprising sulfasalazine and apharmaceutically acceptable excipient, wherein the dose of thepharmaceutical composition is sufficient to produce a plasma level ofsulfasalazine in the patient of at least 8 μg/ml for the entire dosinginterval. For the purposes of this application, the condition “for theentire dosing interval” will be considered to be met if the level of thesulfasalazine is at or above the designated level at the end of thedosing interval (but before any next administration of thesulfasalazine). In certain of those embodiments, the dose of thepharmaceutical composition is sufficient to produce a plasma level ofsulfasalazine in the patient of at least 10 μg/ml for the entire dosinginterval. In certain other embodiments, the dose of the pharmaceuticalcomposition is sufficient to produce a plasma level of sulfasalazine inthe patient of at least 16 μg/ml for the entire dosing interval. Incertain embodiments, the present invention provides methods for treatinga patient with P-MS comprising administering to the patient apharmaceutical composition comprising sulfasalazine and apharmaceutically acceptable excipient, wherein the dose of thepharmaceutical composition is sufficient to produce a plasma level ofsulfasalazine in the patient of between about 8 μg/ml and about 30μg/ml, inclusive, for the entire dosing interval. In certain of thoseembodiments, the dose of the pharmaceutical composition is sufficient toproduce a plasma level of sulfasalazine in the patient of between about10 μg/ml and about 30 μg/ml, inclusive, for the entire dosing interval.In certain other embodiments, the dose of the pharmaceutical compositionis sufficient to produce a plasma level of sulfasalazine in the patientof between about 8 μg/ml and about 16 μg/ml, inclusive, for the entiredosing interval. In certain other embodiments, the dose of thepharmaceutical composition is sufficient to produce a plasma level ofsulfasalazine in the patient of between about 8 μg/ml and about 12μg/ml, inclusive, for the entire dosing interval. In certain embodimentsof this paragraph, the sulfasalazine is in an essentially amorphousform.

One way to increase plasma levels of sulfasalazine is to administerhigher daily doses of the standard formulation of sulfasalazine topatients. Previous work has demonstrated that, in humans, plasma levelsof sulfasalazine are proportional to the oral dose, e.g. Khan et al, Gut21:232-240 (1980). In certain embodiments, the present inventionprovides methods for treating a patient with P-MS comprising orallyadministering to the patient a pharmaceutical composition comprisingsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is a standard formulation of sulfasalazineand the total daily dose of sulfasalazine is between about 2.5 grams andabout 8 grams, inclusive. In certain of those embodiments, the totaldaily dose of sulfasalazine is between about 3 grams and about 5 grams,inclusive. In certain embodiments, the present invention providesmethods for treating a patient with P-MS comprising orally administeringto the patient a pharmaceutical composition comprising sulfasalazine anda pharmaceutically acceptable excipient, wherein the pharmaceuticalcomposition is a standard formulation of sulfasalazine and the totaldaily dose of sulfasalazine is about 3 grams, about 4 grams, or about 5grams.

In certain embodiments, the present invention provides methods fortreating a patient with P-MS comprising orally administering to thepatient a pharmaceutical composition comprising sulfasalazine and apharmaceutically acceptable excipient, wherein (a) the pharmaceuticalcomposition is a standard formulation of sulfasalazine, (b) the dose ateach dosing time point is not more than about 4 grams of sulfasalazine,(c) there are at least two dosing time points in a day, and (d) thetotal daily dose is between about 2.5 grams and about 8 grams,inclusive. In certain of those embodiments, there are two dosing timepoints in a day. In certain of those embodiments, there are three dosingtime points in a day. In certain other embodiments, there are fourdosing time points in a day.

In certain embodiments, methods are provided for treating P-MS in apatient comprising orally administering to the patient a pharmaceuticalcomposition comprising sulfasalazine and a pharmaceutically acceptableexcipient, wherein the pharmaceutical composition is a standardformulation of sulfasalazine and the dose of sulfasalazine is betweenabout 2.5 grams and about 6 grams, inclusive, and is administered once aday. In certain of those embodiments, the dose of sulfasalazine isbetween about 3 grams and about 5 grams, inclusive, and is administeredonce a day. In certain of those embodiments, the dose of sulfasalazineis about 3 grams, about 4 grams, or about 5 grams and is administeredonce a day.

Treatment of Diseases and Disorders Other than NeurodegenerativeDiseases and Disorders

In other embodiments, there is provided a method for treating diseaseswhere sulfasalazine is currently used clinically and is believed to actsystemically, including rheumatoid arthritis and ankylosing spondylitis,wherein such method comprises administering a composition of theinvention comprising sulfasalazine in which the solubility and/or thebioavailability of the sulfasalazine is increased. In rheumatoidarthritis, the typical maintenance dose of sulfasalazine is 2 g/day.Higher doses have been shown to result in greater efficacy, but,unfortunately, the higher doses of sulfasalazine also result in a higherincidence of toxicity, e.g. Khan et al, Gut 21:232-240 (1980). Byincreasing the solubility and/or the bioavailability of thesulfasalazine, the present invention provides a method of increasing thetherapeutic dose of sulfasalazine without an increase in the toxicity.

The present invention provides methods for treating a patient withrheumatoid arthritis and/or ankylosing spondylitis comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the sulfasalazine is in an essentiallyamorphous form. In certain of those embodiments, the pharmaceuticalcomposition is one of the pharmaceutical compositions provided by thepresent application. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform. In certain embodiments, the pharmaceutical composition comprises apharmaceutically acceptable polymer. In certain of those embodiments,the pharmaceutically acceptable polymer is a pyrrolidone polymer. Incertain of those embodiments, the pyrrolidone polymer is selected fromthe group consisting of polyvinylpyrrolidone, vinylpyrrolidone-vinylacetate and crospovidone. In certain embodiments, the pyrrolidonepolymer is PVP VA64. In certain of those embodiments, the ratio of thesulfasalazine to PVP VA64 in the pharmaceutical composition is about20:80 wt/wt to 30:70 wt/wt. In certain embodiments, the ratio ofsulfasalazine to PVP VA64 is about 25:75 wt/wt.

In certain embodiments, there are provided methods for treatingrheumatoid arthritis and/or ankylosing spondylitis in a patientcomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is at least 25%, at least 50%, at least 100%, at least 150%, atleast 200%, at least 250% or at least 300% higher than the plasma levelof sulfasalazine thirty minutes after administration of the same doselevel of crystalline sulfasalazine to a rat as determined by the methodof Example 10. In certain embodiments, there are provided methods fortreating rheumatoid arthritis and/or ankylosing spondylitis in a patientcomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that oral administrationof the formulated pharmaceutical composition to a rat results in aplasma level of sulfasalazine thirty minutes after such administrationthat is about 25%, about 50%, about 100%, about 150%, about 200%, about250% or about 300% higher than the plasma level of sulfasalazine thirtyminutes after administration of the same dose level of crystallinesulfasalazine to a rat as determined by the method of Example 10. Incertain embodiments, the pharmaceutical composition is formulated suchthat oral administration of the formulated pharmaceutical composition toa rat results in a plasma level of sulfasalazine thirty minutes aftersuch administration that is between about 25% and about 500%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain of thoseembodiments, the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 75% and about 300%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain other of thoseembodiments, the pharmaceutical composition is formulated such that oraladministration of the formulated pharmaceutical composition to a ratresults in a plasma level of sulfasalazine thirty minutes after suchadministration that is between about 100% and about 200%, inclusive,higher than the plasma level of sulfasalazine thirty minutes afteradministration of the same dose level of crystalline sulfasalazine to arat as determined by the method of Example 10. In certain embodimentsdescribed in this paragraph, the pharmaceutical composition is one ofthe pharmaceutical compositions provided by the present application. Incertain of those embodiments, the pharmaceutical composition is in anoral dosage form or in a spray dried dispersion form.

In certain embodiments, methods are provided for treating a patient withrheumatoid arthritis and/or ankylosing spondylitis comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition comprisesPVP VA 64 and the ratio of the sulfasalazine to PVP VA64 in thepharmaceutical composition is about 20:80 wt/wt to 30:70 wt/wt. Incertain of those embodiments, the ratio of sulfasalazine to PVP VA64 isabout 25:75 wt/wt. In certain of the above embodiments, wherein thesulfasalazine dispersed in the polymer is in an essentially amorphousform.

In certain embodiments, methods are provided for treating a patient withrheumatoid arthritis and/or ankylosing spondylitis comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and HPMCAS, whereinthe ratio of the sulfasalazine to HPMCAS in the composition is about20:80 wt/wt to 30:70 wt/wt. In certain of those embodiments, the ratioof sulfasalazine to HPMCAS is about 25:75 wt/wt. In certain of thoseembodiments, the sulfasalazine is dispersed in an essentially amorphousform.

In certain embodiments, the application provides methods for treatingrheumatoid arthritis and/or ankylosing spondylitis in a patientcomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is at least 500 μg/ml, at least 1200μg/ml or at least 2300 μg/ml at a pH of 5.5 determined as in Example 9.In certain embodiments, the application provides methods for treatingrheumatoid arthritis and/or ankylosing spondylitis in a patientcomprising orally administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount ofsulfasalazine and a pharmaceutically acceptable excipient, wherein thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is about 500 μg/ml, about 1200 μg/ml orabout 2300 μg/ml at a pH of 5.5 as determined in Example 9. In certainembodiments, the application provides methods for treating rheumatoidarthritis and/or ankylosing spondylitis in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 11,500 μg/ml, inclusive, at a pH of5.5 determined as in Example 9. In certain of those embodiments, thepharmaceutical composition is formulated such that the in vitrosolubility of the sulfasalazine is between about 2300 μg/ml and about11,500 μg/ml, inclusive, at a pH of 5.5 determined as in Example 9. Incertain other of those embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine isbetween about 500 μg/ml and about 2500 μg/ml, inclusive, at a pH of 5.5determined as in Example 9. In certain embodiments described in thisparagraph, the pharmaceutical composition is one of the pharmaceuticalcompositions provided by the present application. In certain of thoseembodiments, the pharmaceutical composition is in an oral dosage form orin a spray dried dispersion form.

In certain embodiments, methods are provided for treating rheumatoidarthritis and/or ankylosing spondylitis in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined in Example 9 is at least 2 times higher than thein vitro solubility of crystalline sulfasalazine at a pH of 5.5 by AUCanalysis. In certain of those embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined in Example 9 is at least 5times or at least 8.8 times higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5 by AUC analysis. In certainembodiments, methods are provided for treating rheumatoid arthritisand/or ankylosing spondylitis in a patient comprising orallyadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of sulfasalazine and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined in Example 9 is about 2 times, about 5 times, orabout 8.8 times higher than the in vitro solubility of crystallinesulfasalazine at a pH of 5.5 by AUC analysis. In certain embodiments,methods are provided for treating rheumatoid arthritis and/or ankylosingspondylitis in a patient comprising orally administering to the patienta pharmaceutical composition comprising a therapeutically effectiveamount of sulfasalazine and a pharmaceutically acceptable excipient,wherein the pharmaceutical composition is formulated such that the invitro solubility of the sulfasalazine at a pH of 5.5 as determined inExample 9 is between about 2 times and about 44 times, inclusive, higherthan the in vitro solubility of crystalline sulfasalazine at a pH of 5.5by AUC analysis. In certain of those embodiments, the pharmaceuticalcomposition is formulated such that the in vitro solubility of thesulfasalazine at a pH of 5.5 as determined in Example 9 is between about2 times and about 8.8 times, inclusive, higher than the in vitrosolubility of crystalline sulfasalazine at a pH of 5.5 by AUC analysis.In certain of those embodiments, the pharmaceutical composition isformulated such that the in vitro solubility of the sulfasalazine at apH of 5.5 as determined in Example 9 is between about 8.8 times andabout 44 times, inclusive, higher than the in vitro solubility ofcrystalline sulfasalazine at a pH of 5.5 by AUC analysis. In certainembodiments described in this paragraph, the pharmaceutical compositionis one of the pharmaceutical compositions provided by the presentapplication. In certain of those embodiments, the pharmaceuticalcomposition is in an oral dosage form or in a spray dried dispersionform.

In other embodiments, there is provided a method for increasing thebioavailability of sulfasalazine in a mammal, the method comprisesorally administering a spray dried dispersion composition comprising atherapeutically effective amount of sulfasalazine and a pyrrolidonepolymer, wherein the sulfasalazine dispersed in the polymer is in anessentially amorphous form. In certain of those embodiments, thesulfasalazine is in an amorphous form. In certain of those embodiments,the spray dried dispersion further comprises a pharmaceuticallyacceptable excipient.

Other System X_(c) ⁻ Inhibitors

In some embodiments, methods for treating a patient with aneurodegenerative disease or disorder comprising administering to thepatient an effective amount of an inhibitor of system x_(c) ⁻ other thansulfasalazine are provided. In certain of those embodiments, the systemx_(c) ⁻ inhibitor is selected from (S)-4-carboxyphenylglycine,2-hydroxy-5-((4-(N-pyridin-2-ylsulfamoyl)phenyl)ethynyl)benzoic acid,aminoadipate (AAA),4-(1-(2-(3,5-bis(trifluoromethyl)phenyl)hydrazono)ethyl)-5-(4(trifluoromethyl)benzyl)isoxazole-3-carboxylic acid,5-benzyl-4-(1-(2-(3,5-bis(trifluoro-methyl)phenyl)hydrazono)ethyl)isoxazole-3-carboxylicacid and2-hydroxy-5-[2-[4-[(3-methylpyridin-2-yl)sulfamoyl]phenyl]ethynyl]benzoic acid.

The following embodiments, aspects and variations thereof are exemplaryand illustrative are not intended to be limiting in scope.

DESCRIPTIONS OF THE FIGURES

FIG. 1 shows a representative Kaplan-Meier absolute survival curves inSOD1^(G93A) mice (herein after “SOD1 mice”). The vehicle-treated (CTRL)and sulfasalazine-treated (DRUG) cohorts are plotted in gray and black,respectively.

FIG. 2 is representative of histograms showing the distribution oflifespan in vehicle and sulfasalazine treated mice after onset ofdefinitive neurological disease.

FIG. 3 shows a representative graph of the percent change in lifespanafter onset of definitive neurological disease in SOD1 mice treated withriluzole, ibuprofen, MR1 and sulfasalazine.

FIG. 4 shows a representative samples from Day 100 mice stained for xCTprotein expression (brown).

FIG. 5 is representative of an area fraction analysis of xCT expressionin the ventral horn of the cervical and lumbar regions of the spinalcord in day 85 and day 100 mice. The symbol ‘*’ indicates the indicatedmeasurement between groups reached a statistical significance of p<0.05.

FIG. 6 shows a representative graph of an area fraction analysiscomparing xCT expression in day 85 and day 100 mice. The y-axisquantifies the xCT expression in the ventral horn of the combinedcervical, thoracic and lumbar spinal cord regions in vehicle treatedSOD1 mice and wild-type mice. The symbol ‘*’ indicates the indicatedmeasurement between groups reached a statistical significance of p<0.05.

FIG. 7 shows representative images from the ventral horn of the spinalcord from Day 85 mice stained for microglial activation using anti-F4/80antibody. Activated microglia are stained brown.

FIG. 8 shows representative samples from Day 100 mice stained forastrocyte activation using anti-GFAP antibody. Activated astrocytes arestained brown.

FIG. 9 shows area fraction quantitation of the activated astrocytes andmicroglial cells in the ventral horn from the cervical and lumbarregions in day 85 mice. Images were analyzed in a blinded fashion andmean area fraction occupied by stain was tabulated. The symbols ‘*’ and‘**’ indicate the indicated measurement between groups reached astatistical significance of p<0.05 and p<0.01, respectively.

FIG. 10 shows area fraction quantitation of the activated astrocytes andmicroglial cells in the ventral horn from the cervical and lumbarregions in day 100 mice. The symbols ‘*’, ‘**’ and ‘***’ indicate themeasurement between groups reached a statistical significance of p<0.05,p<0.01 and p<0.001, respectively.

FIG. 11 shows a representative graph of sulfasalazine concentration inthe spinal cord of SOD1 mice versus time.

FIG. 12 is a graphical representation of the in vitro solubility ofsulfasalazine as a function of pH.

FIG. 13 is a representative graph of results from powder X-raydiffraction (PXRD) analysis of various sulfasalazine formulations.

FIG. 14 is a representative graph of results from modulated differentialscanning calorimetry (mDSC) analysis of sulfasalazine compositions. Theresulting glass-transition temperature (Tg) curve is used to determinethe homogeneity of the composition.

FIG. 15 is a representative graph of results measuring solubility ofsulfasalazine preparations at gastric buffer (GB) and intestinal buffer(IB) of the sulfasalazine formulations.

FIG. 16 is a representative graph of results showing an increase in oralbioavailability of sulfasalazine in a Sprague-Dawley rats followingreformulation.

In addition to the exemplary embodiments, aspects and variationsdescribed above, further embodiments, aspects and variations will becomeapparent by reference to the drawings and figures and by examination ofthe following descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless specifically noted otherwise herein, the definitions of the termsused are standard definitions used in the art of organic synthesis andpharmaceutical sciences. Exemplary embodiments, aspects and variationsare illustrated in the figures and drawings, and it is intended that theembodiments, aspects and variations, and the figures and drawingsdisclosed herein are to be considered illustrative and not limiting.

As used herein, “neurodegenerative disease or disorder” means diseasesof the nervous system that are caused, at least in part, by excessiveglutamate signaling. Examples of neurodegenerative diseases whereanti-glutamatergics are used clinically include Parkinson's disease(Amantadine and Budipine), Alzheimer's disease (Memantine), neuropathicpain (Topamax, Pregabalin), epilepsy (Carbamazepine, Lamictal, Keppra),and ALS (Rilutek). Anti-glutamatergic agents are also being investigatedfor use in traumatic brain injury, Huntington's disease, ischemic strokeand multiple sclerosis.

“Amorphous” refers to a solid state form of a compound (e.g.,sulfasalazine) that is non-crystalline, having no or substantially nomolecular lattice structure, wherein the three dimensional structurepositions of the molecules relative to one another are essentiallyrandom. See for example, Hancock et al. “Characteristics andsignificance of the amorphous state in pharmaceutical systems” J. Pharm.Sci. Vol. 86, pp. 1-12 (1997). As a result, an amorphous material willhave liquid-like short range order and, when examined by X-raydiffraction, will generally produce broad, diffuse scattering and willresult in peak intensity sometimes centered on one or more broad bands(known as an amorphous halo). Thus, PXRD analysis of an amorphousmaterial will provide a 2-theta pattern with one or more broad bandswith no distinctive peaks; unlike the patterns of a crystalline solidmaterial. As used herein, “essentially amorphous” means that thecompound in the material is in at least 80% amorphous form (that is, nomore than 20% crystalline compound), which means such material mayexhibit one or more distinctive peaks in a PXRD analysis.

“Bioavailability” refers the percentage of a dose of a drug that entersthe circulation when that dose of the drug is administered orally to ahuman, rodent, or other animal.

“In vitro solubility” in reference to the solubility of sulfasalazinemeans the C_(max) IB at 90 minutes value for the solubility ofsulfasalazine (as exemplified in Table 9) when measured by the methodsof Example 9.

“Standard formulation of sulfasalazine” refers to formulations ofsulfasalazine that are considered to be essentially equivalent toAzulfidine in terms of the bioavailability of the sulfasalazine in theformulation. These formulations include, but are not limited to,Azulfidine®, Azulfidine® EN (enteric coated), Salazopyrin®, Salazopyrin®EN (enteric coated), SULFASALAZINE TABLETS (Watson Laboratories),SULFAZINE© (Vintage Pharmaceuticals, Inc.), Sazo En (WallacePharmaceuticals Ltd.), Salazopyrin EN (Wallace Pharmaceuticals Ltd.),Salazopyrin (Wallace Pharmaceuticals Ltd.), Sazo EC (WallacePharmaceuticals Ltd.), Saaz (IPCA Laboratories Ltd.), Saaz DS (IPCALaboratories Ltd.), Zemosal (Sun Pharmaceutical Industries Ltd.),Colizine (Synmedic Laboratories), Iwata (Cadila Pharmaceuticals Ltd.),and Salazar EC (Cadila Pharmaceuticals Ltd.).

“Dosing interval” in this application means the period of time betweenadministrations of a composition to a patient. For example, if a drug isadministered to a patient every 8 hours, then the dosing interval is the8 hour period that follows the administration of the drug. For thepurposes of this application, the condition “for the entire dosinginterval” will be considered to be met if the level of the sulfasalazineis at or above the designated level at the end of the dosing interval(but before any next administration of the sulfasalazine).

“Excipient” is a material used in the compositions of the presentapplication, andmay be solid, semisolid or liquid materials which serveas vehicles, carriers or medium for the active compound, such assulfasalazine. Typical excipients may be found in Remington: The Scienceand Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott,Williams & Wilkins, Philadelphia, Pa.; Handbook of PharmaceuticalExcipients by Raymond C. Rowe, Paul J. Sheskey, Walter G. Cook andMarian E. Fenton. 7th Edition, Pharmaceutical Press, London, UK and TheUnited States Pharmacopeia and National Formulary (USP-NF), Rockville,Md. Excipients include, but are not limited to, pharmaceuticallyacceptable polymers.

“Pharmaceutically acceptable salts” means salt compositions that isgenerally considered to have the desired pharmacological activity, isconsidered to be safe, non-toxic and is acceptable for veterinary andhuman pharmaceutical applications. Such salts include acid additionsalts formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, and the like; or with organicacids such as acetic acid, propionic acid, hexanoic acid, malonic acid,succinic acid, malic acid, citric acid, gluconic acid, salicylic acidand the like.

“PVP VA64,” as used herein, means vinylpyrrolidone-vinyl acetatecopolymers with the general formula (C₆H₉NO)_(n)×(C₄H₆O₂)_(m). Sourcesof PVP VA64 include, but are not limited to, BASF (Ludwigshafen,Germany) as Kollidon® VA 64 and Shanghai Lite Chemical Technology Co.,Ltd. As Copovidone (PVP/VA64).

“Copolyvidone,” “Crospovidone” or “polyvinylpyrrolidonepolyvinylacetate” is a polyvinylpyrrolidone polyvinylacetate copolymer.

“Polyvinylpyrrolidone” or “PVP” refers to a polymer, either ahomopolymer or copolymer, containing N-vinylpyrrolidone as the monomericunit. Typical PVP polymers are homopolymeric PVPs and the copolymervinyl acetate vinylpyrrolidone. The homopolymeric PVPs are known to thepharmaceutical industry under a variety of designations includingPovidone, Polyvidone, Polyvidonum, Polyvidonum soluble andPoly(l-vinyl-2-pyrrolidone). The copolymer vinyl acetatevinylpyrrolidone is known to the pharmaceutical industry as Copolyvidon,Copolyvidone and Copolyvidonum.

“Progressive multiple sclerosis” or “P-MS” refers to all the sub-typesof Progressive Multiple Sclerosis characterized by chronic accumulationof disability, which are Primary Progressive Multiple Sclerosis (PP-MS),Secondary Progressive Multiple Sclerosis (SP-MS) andProgressive-Relapsing Multiple Sclerosis (PR-MS).

“Therapeutically effective amount” means an amount of sulfasalazine orother active ingredient of the application that elicits any of thetreatment effects listed in the specification. To be clear, when a unitdose of an active ingredient in the present application is administeredin multiple doses a day, the term “therapeutically effective amount”includes unit doses that are themselves sub-therapeutic, but thatcumulatively result in an administered amount that elicits a treatmenteffect.

“Treating” or “treatment” of a disease as used herein means (a)inhibiting or delaying progression of the disease, (b) reducing theextent of the disease, (c) reducing or preventing recurrence of thedisease, and/or (d) curing the disease. Treating or treatment include,but are not limited to, one or more of (1) limiting, inhibiting orreducing the rate of accumulation of disability and/or loss of motorneuron function; (2) delaying the progression of the disease, such asP-MS or ALS; (3) limiting, inhibiting or reducing neuronal dysfunctionand/or muscular atrophy, (4) limiting or arresting its development, (5)relieving the disease, such as P-MS or ALS, i.e., causing the regressionof P-MS or ALS; (6) reducing or preventing the recurrence of theaccumulation of disability and/or the loss of motor neuron function; (7)reducing or preventing the recurrence of neuronal dysfunction and/ormuscular atrophy; (8) palliating the symptoms of the disease, such asP-MS ALS, (9) increase in survival after onset of P-MS or ALS; and/or,(10) attenuation of neuroinflammation.

Therapeutic Compositions

The invention provides pharmaceutical compositions for use in treatingneurodegenerative diseases or disorders. In some embodiments, thepharmaceutical compositions comprising sulfasalazine are formulated suchthat the bioavailability of the sulfasalazine in the administeredpharmaceutical composition is increased in comparison to administrationof crystalline sulfasalazine or the standard formulation ofsulfasalazine.

In one aspect, the pharmaceutical compositions of the invention mayemploy PVP VA64 or polymer compositions that are related to PVP VA64,and such compositions may include polyvinyl pyrrolidone andpolyoxyethylene-polyoxypropylene copolymers (also known as poloxamers).In another aspect, the pharmaceutical compositions of the invention mayalso employ one or more polymer compositions and additives that arerelated to HPMCAS-MG such as hydroxypropyl methyl cellulose acetatesuccinate (the L, M, and H grades, known as AQUAT-L, AQUAT-M and AQUAT-Hgrades), hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalate (CAP), cellulose acetate trimellitate (CAT), celluloseacetate succinate, methylcellulose acetate succinate, carboxymethylethyl cellulose (CMEC), hydroxypropyl methyl cellulose, andhydroxypropyl methyl cellulose acetate.

Pharmaceutically acceptable polymers include, but are not limited to,PVP VA64, polyvinyl pyrrolidone, polyoxyethylene-polyoxypropylenecopolymers (also known as poloxamers), hydroxypropyl methyl celluloseacetate succinate (HPMCAS), hydroxypropyl methyl cellulose phthalate(HPMCP), cellulose acetate phthalate (CAP), cellulose acetatetrimellitate (CAT), hydroxypropyl methyl cellulose acetate phthalate,hydroxypropyl methyl cellulose acetate trimellitate, cellulose acetatesuccinate, methylcellulose acetate succinate, carboxymethyl ethylcellulose (CMEC), hydroxypropyl methyl cellulose, and hydroxypropylmethyl cellulose acetate, hydroxypropyl methyl cellulose acetatesuccinate (the L, M, and H grades, known as AQUAT-L, AQUAT-M and AQUAT-Hgrades).

In one aspect, the pharmaceutical compositions of the invention comprisesulfasalazine and a polymer, wherein the ratio of sulfasalazine topolymer in the composition is about 1:99 wt/wt to 50:50 wt/wt. Inanother aspect, the ratio of sulfasalazine to polymer is about 5:95wt/wt to 45:55 wt/wt, about 10:90 wt/wt to about 40:60 wt/wt, about15:85 wt/wt to about 35:65 wt/wt, or about 20:80 wt/wt to about 30:70wt/wt.

Also provided are pharmaceutical compositions comprisingpharmaceutically acceptable excipients. Such excipients include, but arenot limited to, polyvinylpyrrolidinone, gelatin, hydroxycellulose,acacia, polyethylene glycol, mannitol, sodium chloride, sodium citrate,and pharmaceutically acceptable polymers.

Pharmaceutically acceptable solid or liquid carriers may be added toenhance or stabilize the composition, or to facilitate preparation ofthe composition. Liquid carriers include syrup, peanut oil, olive oil,glycerin, saline, alcohols or water. Solid carriers include starch,lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate orstearic acid, talc, pectin, acacia, agar or gelatin. The carrier mayalso include a sustained release material such as glyceryl monostearateor glyceryl distearate, alone or with a wax. The amount of solid carriervaries but, preferably, will be between about 20 mg to about 1 g perdosage unit. The pharmaceutical preparations are made following theconventional techniques of pharmacy involving milling, mixing,granulation and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. When aliquid carrier is used, the preparation will be in the form of a syrup,elixir, emulsion, or an aqueous or non-aqueous suspension. Such a liquidformulation may be administered directly by mouth or filled into a softgelatin capsule.

In some embodiments, pharmaceutical compositions of the inventioninclude a pharmaceutically acceptable, non-toxic composition formed bythe incorporation of any of the normally employed excipients, such as,for example, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and combinations thereof. Suchcompositions include suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations and the like.

In addition, the compositions can comprise pharmaceutically acceptablecarriers or customary auxiliary substances, such as glidants; wettingagents; emulsifying and suspending agents; preservatives; antioxidants;anti-irritants; chelating agents; coating auxiliaries; emulsionstabilizers; film formers; gel formers; odor masking agents; tastecorrigents; resin; hydrocolloids; solvents; solubilizers; neutralizingagents; diffusion accelerators; pigments; quaternary ammonium compounds;silicone derivatives; spreading auxiliaries; stabilizers; sterilants;suppository bases; tablet auxiliaries, such as binders, fillers,disintegrants or coatings; propellants; drying agents; pacifiers;thickeners; waxes; plasticizers and white mineral oils.

In some embodiments, the pharmaceutical compositions of the inventionare administered in oral dosage form. Oral dosage forms that may be usedin the invention include, but are not limited to, pills, tablets,chewable tablets, capsules, syrups, sustained release formulations, andsuspensions. In some embodiments, where the composition is a pill ortablet, the composition may contain, along with sulfasalazine, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, gelatin, polyvinylpyrolidinecellulose and derivatives thereof,and the like. In other embodiments, tablet forms of the composition mayinclude one or more of lactose, sucrose, mannitol, corn starch, potatostarch, alginic acid, microcrystalline cellulose, acacia, gelatin, guargum, colloidal silicon dioxide, crosscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, preservatives,flavoring agents, pharmaceutically acceptable disintegrating agents,moistening agents and pharmacologically compatible carriers; andcombinations thereof. In other embodiments, formulations suitable fororal administration can consists of liquid suspensions such as aneffective amount of sulfasalazine suspended in diluents such as water,saline, or orange juice; sachets, lozenges and troches, each containinga predetermined amount of sulfasalazine as solids or granules; powders,suspensions in the above appropriate liquid; and suitable emulsions.

In certain embodiments, pharmaceutical compositions containing a soliddispersion of sulfasalazine and at least one polymer are providedwherein the sulfasalazine is present in essentially amorphous form. Inother embodiments, a method for the preparation of amorphoussulfasalazine is provided. One method for producing a solid moleculardispersion of amorphous sulfasalazine provided herein involves solventspray drying. Other techniques that can be used to prepare solidmolecular dispersions of amorphous sulfasalazine include, withoutlimitation: (1) milling; (2) extrusion; (3) melt processes, includinghigh melt-congeal processes and melt-congeal processes; (4) solventmodified fusion; (5) solvent processes, including spray coating,lyophilization, solvent evaporation (e.g., rotary evaporation) andspray-drying; and (6) non-solvent precipitation.

In one aspect, pharmaceutical compositions of the invention areformulated through spray drying. There are various methods for creatingspray dried compositions, e.g., see EP1469830, EP1469833, EP1653928, WO2010/111132, WO 96/09814; WO 97/44013; WO 98/31346; WO 99/66903; WO00/10541; WO 01/13893, WO 2012/031133, WO 2012/031129, and U.S. Pat.Nos. 6,763,607, 6,973,741, 7,780,988, and 8,343,550. In certain of theseembodiments, the volume mean diameter of the spray dried dispersion isless than about 500 micrometers in diameter or less than about 200micrometers or less than about 100 micrometers or less than about 50micrometers or less than about 10 micrometers. In certain embodiments,the pharmaceutical compositions of the invention are formulated asnanoparticles. There are multiple approaches for formulatingpharmaceutical compositions as nanoparticles, e.g., see WO 2009/073215,U.S. Pat. Nos. 8,309,129; 8,034,765 and 5,118,528.

Typical loadings of sulfasalazine in the formulation can range from 1 wt% API to 50 wt % in the compositions, although more likely they willrange from 5 wt % API to 50 wt %, or 10 wt % to 40 wt %. This willdepend on several factors, including (1) the nature of the polymers inthe composition, and (2) the storage stability of the composition (e.g.,its tendency to phase separate). The sulfasalazine prepared and used inthe compositions of the present application may be amorphous. In oneparticular aspect, the PXRD spectrum of the amorphous sulfasalazineshows a 2-theta pattern with a broad band having no distinctive peaks.In another aspect, the sulfasalazine used in the compositions of thepresent application are at least 80% amorphous, 90% amorphous, at least93% amorphous, at least 95% amorphous, at least 97% amorphous, at least98% amorphous, at least 99% amorphous, at least 99.5% amorphous or about100% amorphous. In another aspect, the remaining or the balance of thesulfasalazine used in the compositions are crystalline material,semi-crystalline material or combination of crystalline andsemi-crystalline materials as determined by PXRD.

Also included in the above embodiments, aspects and variations are saltsof sulfasalazine, such as arginate and the like, gluconate, andgalacturonate. Certain of the compounds of the present invention canexist in unsolvated forms as well as solvated forms, including hydratedforms, and are intended to be within the scope of the present invention.Certain of the above compounds may also exist in one or more solid orcrystalline phases or polymorphs, the variable biological activities ofsuch polymorphs or mixtures of such polymorphs are also included in thescope of this invention.

The invention also provides methods for treatment of P-MS, ALS or otherneurodegenerative diseases comprising administering pharmaceuticalcompositions comprising effective amounts of inhibitors of system x_(c)⁻ other than sulfasalazine. In various embodiments, inhibitors of systemx_(c) ⁻ include, but are not limited to (S)-4-carboxyphenylglycine,2-hydroxy-5-((4-(N-pyridin-2-ylsulfamoyl)phenyl)ethynyl)benzoic acid,aminoadipate (AAA),4-(1-(2-(3,5-bis(trifluoromethyl)phenyl)hydrazono)ethyl)-5-(4(trifluoromethyl)benzyl)isoxazole-3-carboxylic acid,5-benzyl-4-(1-(2-(3,5-bis(trifluoro-methyl)phenyl)hydrazono)ethyl)isoxazole-3-carboxylicacid, and2-hydroxy-5-[2-[4-[(3-methylpyridin-2-yl)sulfamoyl]phenyl]ethynyl]benzoic acid. Formulations of pharmaceutical compositions comprisingthese inhibitors can be generated by various methods, including thosedescribed in Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa.

Administration

In various embodiments, pharmaceutical compositions of the invention maybe administered to patients by oral dosing. In certain of thoseembodiments, the pharmaceutical composition comprising sulfasalazine isformulated such that the oral bioavailability of the sulfasalazine ishigher than that of crystalline sulfasalazine or than the currenton-market formulation of sulfasalazine.

In various embodiments, pharmaceutical compositions of the invention maybe administered to a patient by various routes other than oral dosingsuch as, but not limited to, intravenously, intramuscular, buccal andrectal administration. Suitable formulations for each of these methodsof administration may be found in, for example, Remington: The Scienceand Practice of Pharmacy, A. Gennaro, ed., as noted above.

The specific dose of a pharmaceutical composition of the inventionadministered to a patient may be determined considering the variouscircumstances of the patient being treated such as the route ofadministration, the formulation of the pharmaceutical composition, thepatient's medical history, the weight of the patient, the age and sex ofthe patient, and the severity of the condition being treated. In someembodiments, the patient is administered a pharmaceutical compositioncomprising sulfasalazine where in the amount of sulfasalazine is between100 to 20,000 milligrams/dose. In some embodiments, the patient isadministered an amount of sulfasalazine between 200 and 10,000milligrams/dose. In some embodiments, the patient is administered anamount of sulfasalazine between 400 and 4000 milligrams/dose. In someembodiments, the patient is administered a pharmaceutical compositioncomprising sulfasalazine where in the amount of sulfasalazine is between500 and 2,000 milligrams/dose.

The frequency of administration of a pharmaceutical composition of theinvention to a patient may be determined considering the variouscircumstances of the patient being treated such as the route ofadministration, the formulation of the pharmaceutical composition, thepatient's medical history, the weight of the patient, the age and sex ofthe patient, the rate of disease progression and the severity of thecondition being treated. In some embodiments, the patient isadministered a dose of the pharmaceutical composition more than once. Insome embodiments, the patient is administered a dose of thepharmaceutical composition once a day. In some embodiments, the patientis administered a dose of the pharmaceutical composition comprisingsulfasalazine twice a day, three times a day, or four times a day. Insome embodiments, a patient is administered a dose of the pharmaceuticalcomposition of the invention less frequently than once a day, e.g., onceevery two days or once a week.

The length of treatment by the methods of the invention may bedetermined considering the various circumstances of the patient beingtreated such as the patient's medical history, the weight of thepatient, the age and sex of the patient, the rate of disease progressionand the severity of the condition being treated. In some embodiments,the patient is treated for the rest of their lifetime. In someembodiments, the patient is treated for as long as the disease isactive. In some embodiments, the patient is treated for less than onemonth. In some embodiments, the patient is treated for more than onemonth, e.g., for one year.

In some embodiments, pharmaceutical compositions of the invention areadministered to a patient in combination with one or more other drugcompositions. Such one or more other drug compositions may beadministered concurrently with pharmaceutical compositions of theinvention or may be administered at separate times. In certainembodiments, the one or more other drug compositions are formulated intopharmaceutical compositions of the invention. In other embodiments, theone or more drug composition and the pharmaceutical composition of theinvention are administered as separate compositions. In someembodiments, Mitoxantrone, Gilenya, Masitinib, Siponimod, Tcelna,Tecfidera, Lemtrada, Laquinimod, Daclizumab, Ocrelizumab, Cladribine,Daclizumab, Tysabri, Campath, Rituximab, Fingolimod, Azathioprine orIbudilast is administered in combination with a pharmaceuticalcomposition of the invention to patients with P-MS. In certain of thoseembodiments, Mitoxantrone, Gilenya, Masitinib, Siponimod, Tcelna,Tecfidera, Lemtrada, Laquinimod, Daclizumab, Ocrelizumab, Cladribine,Daclizumab, Tysabri, Campath, Rituximab, Fingolimod, Azathioprine orIbudilast is administered in combination with a pharmaceuticalcomposition of the invention comprising sulfasalazine. In certainembodiments, Mitoxantrone, Gilenya, Masitinib, Siponimod, Tcelna,Tecfidera, Lemtrada, Laquinimod, Daclizumab, Ocrelizumab, Cladribine,Daclizumab, Tysabri, Campath, Rituximab, Fingolimod, Azathioprine orIbudilast is administered to a patients with P-MS in combination with apharmaceutical composition comprising sulfasalazine and PVP VA64,wherein the ratio of the sulfasalazine to PVP VA64 in the composition isabout 20:80 wt/wt to 30:70 wt/wt. In some embodiments, riluzole isadministered in combination with a pharmaceutical composition of theinvention to patients with ALS. In certain of those embodiments,riluzole is administered in combination with a pharmaceuticalcomposition of the invention comprising sulfasalazine. In certainembodiments, riluzole is administered to a patients with ALS incombination with a pharmaceutical composition comprising sulfasalazineand PVP VA64, wherein the ratio of the sulfasalazine to PVP VA64 in thecomposition is about 20:80 wt/wt to 30:70 wt/wt.

EXAMPLES

The following examples below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used but some experimentalerrors and deviations should be accounted for. Unless otherwiseindicated, parts and percentages are by weight, temperature in degreesCelsius (° C.) and pressure is at or near atmosphere. These examples maybe employed for the preparation of the compositions and formulation ofthe present application.

Example 1 Treatment with Sulfasalazine Increases Absolute Survival andIncreases the Lifespan of SOD1 Mice after Onset of DefinitiveNeurological Disease

The following experiments demonstrate that treatment with sulfasalazine:(1) increased the absolute lifespan of SOD1 mice, and (2) extendedlifespan of SOD1 mice after onset of definitive neurological disease.This latter survival parameter is relevant to human patients, whotypically will not begin therapy until after definitive diagnosis ofALS.

High-copy SOD1^(G93A) transgenic mice were derived from theB6SJL-TgN(SOD1G93A)1Gur strain, obtained from The Jackson Laboratory(Bar Harbor, Me.) and originally produced by Gurney, e.g. Gurney et al.,Science 264: 1772-1775 (1994). Animal experiments with the SOD1 modelwere performed at ALS Therapy Development Institute (herein “ALS-TDI”;Cambridge, Mass.). All mice were genotyped to verify copy number of theSOD1 transgene. Animal handling and study protocols were as previouslydescribed by ALS-TDI, e.g. Scott et al., Amyotroph. Lateral Scler. 9:4-15 (2008).

Groups were balanced with respect to gender and body weight withingender. In addition, groups were age-matched and littermate-matched.Each male and female in the drug treatment group had a correspondingmale and female littermate in the vehicle control group. A total of 59mice were used in the study, divided into 2 cohorts as shown in Table 1.Each cohort of was balanced between males and females.

TABLE 1 Cohorts used in the survival study Cohort Genotype TreatmentMale/Female 1 (n = 32) SOD1 Vehicle Control 16/16 2 (n = 27) SOD1Sulfasalazine (Drug Treatment) 14/13

Starting at an age of 50 days, mice were administered sulfasalazine orsaline two times per day (8 hours apart), 7 days per week at a dose of200 mg/kg. Sulfasalazine was prepared by weighing 100 mg of compoundinto a 50 mL corning tube. 5 mL of 0.1 N NaOH was added and the tubegently sonicated. Approximately 140 μL of 1 N HCl was then added tobring the pH to 8.00. The resulting 20 mg/mL solution was delivered byintraperitoneal injection at 10 ml/kg. Vehicle treated mice wereadministered saline.

Neurological scores were assessed daily from day 50 for both hind legs.The neurological score was based on a scale of 0 to 4. Criteria used toassign each score level are from Scott et al., Amyotroph. Lateral Scler.9: 4-15 (2008) and are described in Table 2.

TABLE 2 Criteria for assigning neurological scores Score Criteria 0 NoALS symptomology. Full extension of hind legs away from lateral midlinewhen mouse is suspended by its tail, and mouse can hold this for 2seconds, suspended 2-3 times. 1 Initial Pre-ALS symptomology. Collapseor partial collapse of leg extension towards lateral midline (weakness)or trembling of hind legs during tail suspension. 2 Definitiveneurological disease. Toes curl under at least twice during walking 2paper towel lengths (≈12 inches), or any part of foot is dragging alongcage bottom/table. 3 Advanced disease. Rigid paralysis or minimal jointmovement, foot not being used for forward motion. 4 End stage. Mousecannot right itself within 30 seconds from either side.

The date of definitive neurological disease was the day that the mousefirst scored a “2” on the Neurological Score. Upon reaching a score ofNeurological Score of “4”, mice were euthanized and the date of deathwas recorded.

Sulfasalazine had no statistical effect on time of disease onset.Treatment with sulfasalazine increased median absolute survival of theSOD1 mice by 3.5 days, with a p-value of p=0.07 using the Coxproportional hazard likelihood ratio (FIG. 1). While the effect ofsulfasalazine on absolute survival is modest, it is 68% greater thanriluzole, the only approved therapy for ALS, when tested in the sameSOD1 model under similar conditions (sulfasalazine, 2.7% increasedabsolute lifespan vs. riluzole, 1.6% increased absolute lifespan; seee.g. Lincecum et al., Supplementary Material, Nat. Genetics 42: 392-411(2010)).

Sulfasalazine had a much stronger effect on survival after onset ondefinitive neurological disease. Survival after onset of definitiveneurological disease is defined as the total number of days the micelived after reaching definitive neurological disease (Neurological Scoreof 2) and before death (Neurological Score of 4). Survival after onsetof definitive neurological disease was analyzed in two ways. The firstanalysis used the mean ages of definitive disease onset and death tocalculate the mean lifespan of the SOD1 mice after disease onset, withand without sulfasalazine treatment. The mean lifespan of the SOD1 miceafter onset of definitive neurological disease is shown in Table 3. Ahistogram plot of the survival of the SOD1 mice treated with vehicle andsulfasalazine following onset of definitive neurological disease isshown in FIG. 2. These analyses showed that sulfasalazine treated micelived, on average, 39% longer than vehicle treated mice after onset ofdefinitive neurological disease (p=0.02, t-test with Welch's correctionfor unequal variances). The 95% confidence interval ranged from alifespan increase of 21% to 52% compared to the untreated mice.

TABLE 3 Mean lifespan after onset of definitive neurological disease.Mean Day of Total Days of Definitive Mean Survival after Lower 95% Upper95% Neurological Day of Onset of Defini- Confidence Confidence GroupDisease Death tive Disease Interval Interval Vehicle 116.4 128.9 12.510.73 14.27 Sulfasalazine 115.2 132.6 17.4 12.94 21.73 Absolute Change−1.2 3.7 4.9 2.21 7.46 Percent Change −1.0% 2.9% 39.2% 20.6% 52.3%p-value p = 0.64 p = 0.12 p = 0.02

The second method used to analyze the survival data was to compare theexpected and observed number of days the sulfasalazine treated micespent in one of 3 disease categories to determine if there was asignificant difference between the expected and observed values. Thefirst day that a neurological score was determined was day 50 of age;measurements were collected daily afterwards until death.

The three categories were: (1) days spent before definitive neurologicaldisease, e.g. with a neurological score of 0 or 1; (2) days spent duringdefinitive neurological disease, e.g. with a neurological score of 2 or3; and (3) days at death, e.g. with a neurological score of 4. Bydefinition, mice were only in the dead state for 1 day, as they wereeuthanized upon reaching a neurological score of 4; this category wasincluded as a positive control to ensure the integrity of the data andanalysis.

The expected distribution of days the sulfasalazine treated mice spentin each of these three disease categories was calculated from theobserved distribution of the vehicle treated mice, with the nullhypothesis that sulfasalazine treatment had no effect on thedistribution. The observed distribution of sulfasalazine treated mice isbased on daily scoring and is normalized to the number of mice in thegroups.

The results of this analysis are shown in Table 4. The sulfasalazinetreated mice, as a group, survived a total of 112 days longer thanexpected after onset of definitive neurological disease (neurologicalscore of 2 or 3). This result was highly significant by Chi-Squareanalysis, with a p-value of less than 0.0004 by the Wald test and 0.0003by the Likelihood ratio test. There was no significant difference in thetotal days spent in the pre-disease state (neurological score of 0 or 1)or death (neurological score of 4).

TABLE 4 Expected and observed distribution of sulfasalazine neurologicalscores and calculated p-values. Effect Expected Observed Observed Effect(Likelihood Days, Days, Days Minus (Wald Tests)² Ratio Tests)²Measurement Disease Category Vehicle Sulfasalazine¹ SulfasalazineExpected Days Prob > ChiSq Prob > ChiSq Total Days Before definitivedisease 2041 1722 1708 −14 0.0004 0.0003 onset (neurological score = 0or 1) Definitive neurological 357 301 413 112 disease (neurologicalscore = 2 or 3) Death (neurological 32 27 27 0 score = 4) TotalObservations (Days) 2430 2050 2148 98 ¹Expected days for sulfasalazinetreated animals if sulfasalazine has no effect on disease; valuespredicted from vehicle-treated cohort and normalized for number ofanimals in treated cohort. ²Prob > ChiSq is the probability of obtaininga greater Chi-square value by chance alone if treatment has no effect ontime spent within each disease category. All statistics performed usingthe JMP10 statistics program (SAS Institute).

The increased lifespan following onset of definitive neurologicaldisease seen with sulfasalazine was also compared to published resultsof other compounds tested in the SOD1 mouse model. FIG. 3 shows thepercent difference in survival following onset of neurological diseasefor sulfasalazine, two general anti-inflammatory compounds (ibuprofenand MR1, an antibody to CD40L) and riluzole, the only drug currentlyapproved for ALS. Sulfasalazine increased lifespan by 39%, anti-CD40Lincreased lifespan by 9%, riluzole increased lifespan by 1% andibuprofen decreased lifespan after onset of neurological disease by 10%.See, e.g., Shin et al., J. Neurochem. 122: 952-961 (2012); Lincecum etal., Nat. Genetics 42: 392-411 (2010).

This comparison illustrates that the increased lifespan observed withsulfasalazine is significantly larger than is observed with other testedcompounds, including two general anti-inflammatory compounds (ibuprofenand anti-CD40L) and the only approved therapy for ALS (riluzole).

The experiments in the SOD1 animal model of ALS demonstrate that, whilethe effect of sulfasalazine on absolute survival is modest, it wassuperior to riluzole, the only approved therapy for ALS. Importantly,the benefit in survival by sulfasalazine after onset of definitiveneurological disease is large, in terms of absolute size (approximately40%), the statistical significance and when compared to other compounds,including riluzole. It is noteworthy that the entire increase insurvival noticed in the absolute survival analysis (median 3.7 days)occurs after the definitive onset of neurological disease. This resultis consistent with the expression data (FIG. 6, below) that shows xCTexpression escalates with disease progression. Based on the expressionprofile of the target, it is expected that sulfasalazine would havelittle effect on delaying the onset of disease, but would have aprogressively beneficial effect as disease progresses, as was observedin the survival study.

These experiments demonstrate that sulfasalazine has modest efficacy onabsolute survival and strong efficacy on survival after onset ofdefinitive neurological disease in the SOD1 mouse model of ALS. As mostALS patients do not begin therapy until after diagnosis of neurologicaldisease, the latter measurement is especially relevant to the treatmentof human disease.

Example 2 Expression of xCT (SLC7A11) is Elevated in the Spinal Cord ofSOD1 Mice

The following studies used quantitative immunohistochemistry todetermine: (1) if the expression of xCT in the spinal cord was elevatedin SOD1 mice, (2) if so, whether xCT over-expression increased withdisease progression, and (3) whether treatment with sulfasalazineaffected xCT expression in the spinal cord of SOD1 mice.

Two ages of mice were chosen for this analysis: day 85, when SOD1 miceshow no overt sign of the ALS-like symptomology, and day 100, when SOD1mice typically begin displaying the first signs of ALS-likesymptomology, such as partial collapse of leg extension towards lateralmidline (weakness) or trembling of hind legs during a tail suspensiontest. For the immunohistochemical studies, a total of 48 mice weredivided into 6 cohorts of 8 mice each (4 females and 4 males) as shownin Table 5.

TABLE 5 Cohorts used in the Immunohistochemical Studies Cohort (n = 8)Genotype Treatment Age of mouse at sacrifice 1 Wild-type vehicle 85 days2 SOD1 vehicle 85 days 3 SOD1 sulfasalazine 85 days 4 Wild-type vehicle100 days 5 SOD1 vehicle 100 days 6 SOD1 sulfasalazine 100 days

Starting at an age of 50 days, mice were administered sulfasalazine orsaline two times per day (8 hours apart), 7 days per week at a dose of200 mg/kg. Sulfasalazine was prepared by weighing 100 mg of compoundinto a 50 mL corning tube. 5 mL of 0.1 N NaOH was added and the tubegently sonicated. Approximately 140 μL of 1 N HCl was then added tobring the pH to 8.00. The resulting 20 mg/mL solution was delivered byintraperitoneal injection at 10 ml/kg. Vehicle treated mice wereadministered saline.

Mice were sacrificed by CO₂ asphyxiation according to IACUC approvedprotocols. The spinal cord was extruded with cold PBS into a bath ofcold PBS from the vertebral column of mice using an 18 gauge needleinserted in the sacral vertebral column to a friction fit. Uponextrusion, the spinal cord tissue was rinsed and dropped into 4%paraformaldehyde for 24 hours at room temperature (RT, approximately 25°C.). The tissue was then transferred to a 1× phosphate buffered saline(PBS) solution. Samples were then processed by TissueTek processors forparaffin embedding. Spinal cord samples were then embedded in paraffinblocks and oriented for transverse sectioning. Spinal cord samples weresectioned at 10 microns thickness. Three representative sections werecut from lumbar, thoracic and cervical regions of the spinal cord.Samples were pretreated with Pronase for 20 minutes at RT, followed bytreatment with 3% H₂O₂ for 12 minutes at RT. Horse serum was added to 2%and samples incubated for 20 minutes at RT. The samples were thenincubated with the primary antibody (Anti-xCT; purchased from Abcam(Cambridge, Mass.); Catalog #Ab37185; diluted 1:500 in PBS) overnight at4° C. The secondary antibody (Biotin labeled goat anti-rabbit IgG; 1:500dilution in PBS) was then added and the reaction incubated overnight atRT. Reaction products were developed using the Vector ABC system (VectorLabs, Burlingame, Calif.) using avidin-conjugated horseradishperoxidase. xCT expression was visualized by addition of the chromogenicsubstrate DAB (3,3′-diaminobenzidine) for 10 minutes at RT. Each stainedsection was imaged at objectives: 4×, 10×, 20× and 40×. For eachobjective image, light parameters were optimized and kept consistentacross all sections. For SLC7A11 analysis, all images that were capturedat 20× were then imported into ImageJ freeware (NIH, Bethesda, Md.). Amaximum entropy threshold algorithm was applied to all images in acompletely blinded fashion to filter out all pixels that were notstained as DAB positive. The key parameter measured and reported is areafraction. Area fraction is the proportion of total pixels that are DABpositive in the ventral horn of the spinal cord. All statisticalanalyses were performed using JMP® 7.0, SAS Institute, Inc. Areafraction was compared with respect to treatment using 1-way ANOVAanalysis, with a p-value of 0.05 considered significant.

FIG. 4 shows representative images from day 100 mice. Increasedexpression of xCT is visible in the sections from the SOD1 mice, withand without sulfasalazine treatment.

FIG. 5 shows the quantitation of xCT area fraction for the cervical andlumbar regions of the spinal cord in day 85 and day 100 mice. At day 85,xCT protein levels were elevated in both the cervical and lumbar regionsin SOD1 mice, reaching statistical significance (p<0.05) in the lumbarregion (FIG. 5, panel B). At day 100, xCT protein levels were elevatedin both the cervical and lumbar regions in SOD1 mice, reachingstatistical significance (p<0.05) in both regions (FIG. 5, Panels C andD).

FIG. 6 compares total xCT expression levels in the ventral horn of thespinal cord in day 85 and day 100 mice. For this analysis, values forthe cervical, thoracic and lumbar regions were combined into a singlevalue. In the day 85 SOD1 mice, xCT protein levels were elevated byapproximately 50% in the combined spinal cord sections compared to day85 wild-type mice. In the day 100 SOD1 mice, xCT protein levels wereelevated by approximately 300% across in the combined spinal cordssections compared to day 100 wild-type mice.

These results demonstrate that (1) xCT target expression is elevated inthe diseased tissue—the ventral portion of the spinal cord—of SOD1 mice;(2) expression of xCT escalates significantly during disease progressionin the SOD1 mice (day 85 versus day 100, FIG. 6), and (3) treatment withsulfasalazine did not have a significant effect on xCT expression (FIG.5, Panels A-D). Thus, there does not appear to be a compensatory orrebound effect on xCT levels when it is inhibited by sulfasalazine. Sucha rebound effect could lead to loss of efficacy upon treatment.

The increased expression of xCT observed during disease progression isconsistent with sulfasalazine having greatest efficacy during the laterstages of disease.

Example 3 Sulfasalazine Reduces Levels of Neuroinflammatory Cells in theSpinal Cord of SOD1 Mice

The following experiments employed quantitative immunohistochemistry to:(1) compare the neuroinflammatory cell populations in the spinal cord ofSOD1 mice to the cell populations in wild-type mice, and (2) testwhether the treatment with sulfasalazine decreases neuroinflammatorycell populations in the spinal cord of SOD1 mice.

The same test mice, spinal cord preparations and methods of analysisused in the neuroinflammatory study were identical to those used in thexCT quantitation study. Two neuroinflammatory cell populations werequantitated: (1) activated microglial cells using an antibody to theF4/80 antigen, and (2) activated astrocytes, using an antibody to theGFAP antigen. For each objective image, light parameters were optimizedand kept consistent across all sections. Images that were captured at20× were then imported into ImageJ freeware (NIH, Bethesda, Md.). Amaximum entropy threshold algorithm was applied to all images in acompletely blinded fashion to filter out all pixels that were notstained as DAB positive. 20× images were analyzed in a blinded fashionand mean area fraction occupied by stain was tabulated. Levels ofneuroinflammation in the spinal cord were assessed by measuring the areafraction of the area of the ventral horn in the spinal cord occupied byactivated astrocytes or microglia. Area fraction was compared withrespect to treatment using 1-way ANOVA analysis, with a p-value of 0.05considered significant.

For quantitation of microglial activation, samples were pretreated withPronase for 20 minutes at room temperature, followed by treatment with3% H₂O₂ for 12 minutes at room temperature (25° C.). Goat serum wasadded to 2% and samples incubated for 20 minutes at room temperature.The samples were then incubated with the primary antibody (Anti-F4/80)purchased from Serotec (Catalog # MCA497R; Oxford, United Kingdom),diluted 1:250 in PBS overnight at 4° C. The secondary antibody (Biotinlabeled goat anti-rabbit IgG; 1:250 dilution in PBS) was then added andthe reaction incubated 1 hour at room temperature. Reaction productswere developed using the Vector ABC system (Vector Labs, Burlingame,Calif.) using avidin-conjugated horseradish peroxidase (45 minutes atroom temperature). Activated microglia were visualized by addition ofthe chromogenic substrate DAB (3,3′-diaminobenzidine) for 6 minutes atroom temperature.

For quantitation of astrocyte activation, samples were pretreated withheated citrate buffer for 20 minutes, followed by treatment with 3% H₂O₂for 12 minutes at room temperature. Horse serum was added to 2% andsamples incubated for 20 minutes at room temperature. The samples werethen incubated with the primary antibody (Anti-GFAP) purchased fromAbcam (Catalog # Ab10062; Cambridge, Mass.), diluted 1:1000 in PBSovernight at 4° C. Reaction products were developed using the VectorImmPress system (Vector Labs, Burlingame, Calif.) using an anti-mouseIgG-conjugated horseradish peroxidase. Activated astrocytes werevisualized by addition of the chromogenic substrate DAB(3,3′-diaminobenzidine) for 90 seconds at room temperature.

FIG. 7 shows representative images from tissue from day 85 mice stainedfor activated microglia. FIG. 8 shows representative images from tissuefrom day 100 mice stained for activated astrocytes.

FIG. 9 shows area fraction quantitation of the activated astrocytes andmicroglial cells in the ventral horn from the cervical and lumbarregions of the spinal cord in day 85 mice. A strong trend towardastrocyte activation was observed in diseased mice (SOD1) compared tonon-diseased mice (WT) in the lumbar region (FIG. 9, Panel B).Sulfasalazine treatment significantly lowered astrocyte activation inthe lumbar region (FIG. 9, Panel B). Astrocyte activation was notelevated in the cervical region in SOD1 mice vs. WT mice (FIG. 9, PanelA).

In day 85 mice, increased microglial activation was observed in diseasedmice (SOD1) compared to non-diseased mice (WT) in both the cervicalregion (FIG. 9, Panel C) and in the lumbar region (FIG. 9, Panel D),although activation in the lumbar region did not reach statisticalsignificance. Sulfasalazine treatment significantly decreased microglialactivation in the cervical region of SOD1 mice (FIG. 9, Panel C) andalso reduced microglial activation in the lumbar region in SOD1 mice,although this effect did not reach statistical significance.

These results demonstrate that in day 85 SOD1 mice: (1) increased levelsof neuroinflammatory cells (activated astrocytes and microglia) arepresent in the spinal cord before ALS-like symptomology is observed, and(2) treatment with sulfasalazine lowers the overall levels ofneuroinflammatory cells (activated astrocytes and/or microglia) in boththe cervical and lumbar regions of the spinal cord.

FIG. 10 shows area fraction quantitation of the activated astrocytes andmicroglial cells in the ventral horn from the cervical and lumbarregions of the spinal cord in day 100 mice. Significantly increasedastrocyte activation was observed in diseased mice (SOD1) compared tonon-diseased mice (WT) in the cervical region (FIG. 10, Panel A).Sulfasalazine treatment significantly lowered astrocyte activation inthe cervical region (FIG. 10, Panel A). In the lumbar region, there wasa trend towards increased astrocyte activation in the lumber region, butit was not statistically significant. Sulfasalazine treatment did noteffect astrocyte activation in the lumbar region (Panel B).

At day 100, increased microglial activation was observed in diseasedmice (SOD1) compared to non-diseased mice (WT) in the cervical region(FIG. 10, Panel C) and in the lumbar region (FIG. 10, Panel D), althoughactivation in the lumbar region did not reach statistical significance.Sulfasalazine treatment resulted in a trend towards decreased microglialactivation in the cervical region (FIG. 10, Panel C) and did not affectsuch activation in the lumbar region (FIG. 10, Panel D).

These results demonstrate that in Day 100 SOD1 mice: (1) increasedlevels of neuroinflammatory cells (activated astrocytes and microglia)are present in the spinal cord, in particular the cervical region, and(2) treatment with sulfasalazine lowers the overall levels ofneuroinflammatory cells in the cervical region of the spinal cord.

Table 6 contains a summary of all the data from the neuroinflammationexperiment presented in tabular format. The changes in area fractionstaining in the cervical, thoracic, and lumbar regions of the spinalcord, as well as the combined changes across the whole spinal cord (sumof cervical, thoracic and lumbar regions) are scored for the followinggroup comparisons:

(1) Whether increased activation of microglia and astrocytes wasobserved in diseased (SOD1, vehicle treated) mice vs. non-diseased(wild-type) mice (Column 4). In a total of 16 measurements, evidence forastrocyte and/or microglial activation was observed 14 times, reachingstatistical significance 5 times; and

(2) Whether treatment with sulfasalazine decreased activation ofmicroglia and astrocytes compared to vehicle treatment in SOD1 mice(Column 5). From the total of 14 tissues that showed activation ofastrocytes and/or microglia in SOD1 mice, sulfasalazine treatment wasobserved to decrease activation 8 times, reaching statisticalsignificance 4 times.

TABLE 6 Summary of Neuroinflammation Data Column 5: Decreased acti-Column 4: vation in sulfas- Increased activation alazine treated in SOD1(vehicle) SOD1 vs. vehicle Tissue Day Cell type vs. WT mice treated SOD1mice Cervical 85 Astrocytes No increase No decrease Cervical 100Astrocytes Strong increase Strong decrease (p < 0.001) (p < 0.01)Thoracic 85 Astrocytes Trend Trend Thoracic 100 Astrocytes Trend Nodecrease Lumbar 85 Astrocytes Trend Decrease (p < 0.05) Lumbar 100Astrocytes Trend No decrease Combined 85 Astrocytes Trend Decrease (p <0.05) Combined 100 Astrocytes Trend Trend Cervical 85 Microglia IncreaseStrong decrease (p < 0.05) (p < 0.01) Cervical 100 Microglia IncreaseTrend (p < 0.05) Thoracic 85 Microglia No increase No decrease Thoracic100 Microglia Trend No decrease Lumbar 85 Microglia Trend Trend Lumbar100 Microglia Trend No decrease Combined 85 Microglia Increase Trend (p< 0.05) Combined 100 Microglia Increase No decrease (p < 0.05)

This experiment establishes that sulfasalazine treatment lowers thelevels of both activated microglial cells and activated astrocytes inthe spinal cord.

The results from the neuroinflammatory study suggest that xCT activityis required for maximum levels of neuroinflammation to occur.

Example 4 Sulfasalazine Reaches Therapeutic Concentrations in the SpinalCord and Spinal Cord Levels are Proportional to Concentrations in thePlasma

The experimental procedures and results provided below demonstrate theexposure and pharmacokinetics of sulfasalazine in the spinal cord andplasma of SOD1 mice.

Study Protocol and Sample Analysis

SOD1 mice were dosed with sulfasalazine at 200 mg/kg intraperitoneallyand spinal cords and plasma (50 μl) harvested at indicated times (n=3mice per time point). The zero time point was taken before drug wasadministered to serve as a negative control for drug quantitation.Analytical methods were developed and performed by MicroConstants (SanDiego, Calif.). Spinal cords and blood plasma samples (50 μl) werehomogenized in 150 μl of phosphate buffer and then extracted by amixture of methylene chloride and MTBE (1:4 dilution). Sample extractswere analyzed and quantitated by high-performance liquid chromatographyusing a BetaMax Acid column maintained at 35° C. The mobile phase wasnebulized using heated nitrogen in a Z-spray source/interface and theionized compositions were detected and identified using a tandemquadrupole mass spectrometer (MS/MS).

Analytical Method Qualification

A reference standard of sulfasalazine (Sigma-Aldrich, Catalog # S0883)was used to generate a standard curve in rat plasma. The assay gave alinear response to concentrations of sulfasalazine from 10 to 20,000ng/ml (Table 7). Dilution controls showed that samples could be dilutedup to 1:100 and give a linear response in the assay. Curve fitting fromthis data generated the following parameters for the equation used tocalculate unknown concentrations:LOG(y)=A+B*LOG(x) where y=peak height ratio and x=concentration  GeneralEquation:

Specific Parameters for sulfasalazine: A=3.06, B=0.976; Correlationcoefficient=1.00

TABLE 7 Standard Curve Values of Sulfasalazine. Standard Concentrations(ng/mL) Analyte 10.0 20.0 50.0 100 250 500 1,000 2,000 5,000 10,00020,000 Sulfasalazine 9.80 9.88 18.5 46.4 109 255 519 1,100 2,150 5,36010,200 17,800 18,100 (measured) Mean (ng/mL) 9.84 18.5 46.4 109 255 5191,100 2,150 5,360 10,200 18,000 Percent standard −1.60 −7.50 −7.20 9.002.00 3.80 10.0 7.50 7.20 2.00 −10.0 deviation

Separately, an internal standard (deuterated sulfasalazine) was used todetermine compound extraction efficiency from mouse CNS (brain) tissueand plasma. The extraction efficiency of sulfasalazine was determined tobe >98% from mouse brain tissue and plasma.

Table 8 shows the mean values for the concentrations of sulfasalazine inthe spinal cord and in the plasma, and also the standard deviations (SD)of the measurements and the ratio of sulfasalazine in the spinal cord tothe plasma.

TABLE 8 Mean concentrations of sulfasalazine in the CNS (spinal cord)and plasma, standard deviations (SD) and ratios in CNS (spinal cord) toplasma. BQL = below quantitative limit of detection (10 ng/ml) SpinalSpinal Plasma, Plasma, Ratio Time cord, mean cord, SD mean SD (Spinal(min) (ng/g) (ng/g) (ng/ml) (ng/ml) cord/plasma) 0 BQL BQL BQL BQL BQL 517,719 8,081 77,629 6,815 23% 15 10,419 2,479 67,143 3,672 16% 30 10,2132,257 60,235 2,731 17% 45 7,065 1,312 54,687 1,762 13% 60 3,276 61240,354 5,198  8% 120 991 457 19,234 3,279  5% 180 653 119 6,090 994 11%240 20 18 2,580 622  1% 360 BQL BQL 630 121 n/a

FIG. 11 shows the mean logarithmic concentration of sulfasalazine in thespinal cord versus time following administration. Sulfasalazine showedimmediate penetration into the spinal cord, reaching levels ofapproximately 18 μg/gram of tissue within 5 minutes of drugadministration. The levels in the spinal cord ranged from approximately5-17% of the levels in the plasma over the next three hours. The minimumtherapeutic range of sulfasalazine, estimated to be 2 to 2.5 micromolar(equivalent to 800-1,000 ng/ml; assuming a conversion of 1 gram tissue=1ml volume), is shown in the shaded rectangle in the figure. Theseresults show that sulfasalazine reached immediate and therapeutic levelsin the mouse spinal cord. The half-life of sulfasalazine in the spinalcord and plasma was approximately one hour, with the levels in thespinal cord proportional to the levels in the plasma. The observedhalf-life in the spinal cord and plasma is consistent with the reportedhalf-life of sulfasalazine in mouse plasma, see, e.g., Zaher et al.,Mol. Pharmaceutics 3: 55-61 (2005). Therapeutically relevantconcentrations of sulfasalazine were present in the spinal cord forapproximately 2-2.5 hours, corresponding to a concentration ofsulfasalazine in the CNS of approximately 800-1000 ng/g. Correspondingconcentrations of sulfasalazine in the plasma during this time periodranged from approximately 8,000-19,000 ng/ml.

Results of the SOD1 experiments provide strong support thatsulfasalazine has therapeutic applications for ALS, despite the shorthalf-life of sulfasalazine and resulting sub-optimal drug coverage inthese particular studies. Levels of the target—xCT—were elevated indiseased tissue and escalated with disease progression. Treatment withsulfasalazine demonstrated significant efficacy in two importantcomponents of disease: (1) survival after onset of neurological diseaseand, (2) attenuation of neuroinflammation.

Example 5 Determination of Solubility of Crystalline Compound atDifferent pH

The following procedure was used to determine the effect of pH on thesolubility of sulfasalazine in aqueous solutions. A 1.8 mg sample ofsulfasalazine was placed in a microcentrifuge tube. A 0.9 mL of 0.01NHCl was then added to the tube, which was capped and mixed using avortex mixer for 1 minute. The sample in the tube was then centrifugedat 15,800 relative centrifugal force (RCF) for 1 minute. A 50 μL sampleof the liquid was diluted into 250 μL HPLC solvent, and the tube wascapped and vortexed for 20 seconds and allowed to stand undisturbed at37° C. until the next sample was collected. After 30 minutes, a 0.9 mLportion of buffer solution (at twice the concentration of buffer salts)was added to the microcentrifuge tube, and the procedure repeated asdescribed above. Samples were collected at predetermined time intervalsand analyzed by HPLC. The solubility of sulfasalazine as a function ofpH was then determined, as shown in FIG. 12. This data indicates thatcrystalline drug alone may have good bioavailability based on solubilityif the pH of the absorption window is high (pH 6 or above). This dataalso indicates that crystalline drug alone may have poor bioavailabilitybased on solubility if the pH of the absorption window is low (below pH6).

Example 6 Reformulation of Sulfasalazine to Increase OralBioavailability

Novel formulations of sulfasalazine that increase the solubility ofsulfasalazine at enteric pH (i.e., below pH 6) were prepared, includinga formulation of sulfasalazine that increases the oral bioavailabilityof the sulfasalazine by at least three-fold in a rat model.

Preparation of Sulfasalazine Formulations

Sulfasalazine Formulation Exemplar 1: 25% Sulfasalazine:75% HPMCAS SDD

A spray dried dispersion (SDD) of 25 wt % sulfasalazine and 75 wt %HPMCAS (hereafter “25% sulfasalazine:HPMCAS”) was prepared using a spraydrying process as follows. A spray solution was prepared by dissolving100 mg sulfasalazine and 300 mg HPMCAS (Hydroxypropylmethylcelluloseacetate succinate; AQOAT M grade, Shin Etsu, Tokyo, Japan) in 19.6 gm ofsolvent (95/5 w/w tetrahydrofuran/water), to form a spray solutioncontaining 2 wt % solids. This solution was spray dried using asmall-scale spray-dryer, which consisted of an atomizer in the top capof a vertically oriented 11-cm diameter stainless steel pipe. Theatomizer was a two-fluid nozzle, where the atomizing gas was nitrogendelivered to the nozzle at 70° C. at a flow rate of 31 standard L/min(SLPM), and the solution to be spray dried was delivered to the nozzleat room temperature at a flow rate of 1.3 mL/min using a syringe pump.The outlet temperature of the drying gas and evaporated solvent was31.5° C. Filter paper with a supporting screen was clamped to the bottomend of the pipe to collect the solid spray-dried material and allow thenitrogen and evaporated solvent to escape. The resulting spray driedpowder was dried under vacuum overnight, with a yield of 89%.

Sulfasalazine Formulation Exemplar 2: 25% Sulfasalazine:75% PVP VA64 SDD

A spray dried dispersion (SDD) of 25 wt % sulfasalazine and 75 wt % PVPVA64 (hereafter “25% sulfasalazine:PVP VA64”) was prepared using a spraydrying process as follows. The procedure of sulfasalazine formulationExemplar 1 was repeated except that the polymer wasvinylpyrrolidone-vinyl acetate copolymer (PVP VA64, purchased from BASFas Kollidon® VA 64, Ludwigshafen, Germany). The spray drying conditionswere the same as sulfasalazine formulation Exemplar 1. The resultingspray dried powder was dried under vacuum overnight, with a yield of95.7%.

Sulfasalazine Formulation Exemplar 3: 50% Sulfasalazine:50% PVP VA64 SDD

A spray dried dispersion (SDD) of 50 wt % sulfasalazine and 50 wt % PVPVA64 (hereafter “50% sulfasalazine:PVP VA64”) was prepared using a spraydrying process as follows. A spray solution was prepared by dissolving200 mg sulfasalazine and 200 mg PVP VA64 in 19.6 gm of solvent (90/10w/w tetrahydrofuran/water), to form a spray solution containing 2 wt %solids. This solution was spray dried using a small-scale spray-dryer,as described in sulfasalazine formulation Exemplar 1. The resultingspray dried powder was dried under vacuum overnight, with a yield of95.7%.

Example 7 Characterization of the Compositions Showing AmorphousDispersion Using PXRD Analysis

The three exemplar formulations were analyzed by powder X-raydiffraction (PXRD) using an AXS D8 Advance PXRD measuring device(Bruker, Inc. of Madison, Wis.) using the following procedure. Samples(approximately 100 mg) were packed in Lucite sample cups fitted withSi(511) plates as the bottom of the cup to give no background signal.Samples were spun in the φ plane at a rate of 30 rpm to minimize crystalorientation effects. The X-ray source (KCu_(α), λ=1.54 Å) was operatedat a voltage of 45 kV and a current of 40 mA. Data for each sample werecollected over a period of 30 minutes in continuous detector scan modeat a scan speed of 2 seconds/step and a step size of 0.04°/step.Diffractograms were collected over the 2θ range of 4° to 40°. FIG. 13shows the diffraction pattern of the formulations, revealing anamorphous halo, indicating the sulfasalazine in each of the exemplarformulations was essentially amorphous.

Example 8 Characterization of Compositions Showing Homogeneity UsingmDSC Analysis

The exemplar formulations were analyzed using modulated differentialscanning calorimetry (mDSC) as follows. Samples of the formulations(about 2 to 4 mg) were equilibrated at <5% RH overnight in anenvironmental chamber at ambient temperature. The samples were thenloaded into pans and sealed inside the environmental chamber. Thesamples were then analyzed on a Q1000 mDSC (TA Instruments, New Castle,Del.). Samples were scanned over the temperature range of −40° C. to200° C., at a scan rate of 2.5° C./min, and a modulation rate of ±1.5°C./min. The glass-transition temperature (Tg) was calculated based onhalf height. The mDSC results are shown in FIG. 14, and the Tg is alsoreported in Table 9 (data are reported as an average of 3 replicates).In all cases, the dispersions exhibited a single Tg, indicating theactive agent in the dispersion was molecularly dispersed and homogeneousin the SDD.

TABLE 9 Glass transition temperatures of sulfasalazine preparationsSample Tg at < 5% RH (° C.) Formulation Exemplar 1 98.1 ± 0.3 25%Sulfasalazine:HPMCAS SDD Formulation Exemplar 2 110.5 ± 0.03 25%Sulfasalazine:PVP VA64 SDD Formulation Exemplar 3 118.0 ± 0.2  50%Sulfasalazine:PVP VA64 SDD

Example 9 Determination of Solubility of Reformulated Compounds atEnteric pH

The release of sulfasalazine from the dispersions of formulationexemplars 1-3, crystalline sulfasalazine, and amorphous sulfasalazineformulation (made by spray drying) was determined using the followingprocedures. A sample mass of 4.5 mg of the test material was placed in amicrocentrifuge tube. To this was added 0.9 mL of gastric buffer (GB)solution (0.01 N HCl, pH 2). The tubes were vortexed for one minute,then centrifuged for one minute before taking each sample. Samples (theliquid phase) were taken at 5, 15, and 25 minutes. At 30 minutes afterthe start of the test, 0.9 mL of intestinal buffer (IB) solution (aphosphate/citrate buffer at pH 5.5) was added to the tubes (at a doubleconcentration of the buffer salts to result in the desired pH level andbuffer strength). The tubes were vortexed for one minute, thencentrifuged for one minute before taking each sample. Samples were takenat 4, 10, 20, 40, 90 and 1200 minutes after addition of the intestinalbuffer solution. The concentration of sulfasalazine was determined byHPLC as previously described. Table 10 shows the data from thesolubility experiment and FIG. 15 shows the data in graphical format.This data demonstrated that the amorphous sulfasalazine preparation hashigher solubility than the crystalline sulfasalazine, by approximately36%. When the amorphous sulfasalazine was prepared with polymers, thesolubility further increased. The 25% sulfasalazine:HPMCAS-MGformulation had an increase in solubility of approximately 200% comparedto the crystalline sulfasalazine and of approximately 46% over theamorphous sulfasalazine. The 50% sulfasalazine:PVP VA64 polymer had anincrease in solubility of approximately 508% compared to the crystallinesulfasalazine and of approximately 372% over the amorphoussulfasalazine. The 25% sulfasalazine:PVP VA64 polymer had an increase insolubility of approximately 883% compared to the crystallinesulfasalazine and of approximately 647% over the amorphoussulfasalazine.

TABLE 10 Solubility of Compounds in Gastric Buffer and Intestinal BufferCmax IB Cmax IB Ratio of AUC Ratio of AUC Cmax GB Cmax IB (ug/mL)(ug/mL) AUC to cystalline to amorphous Sample (ug/mL) (ug/mL) at 90 minat 1200 min (min * ug/mL) sulfasalazine sulfasalazine Crystallinesulfasalazine 15 282 271 282 22,200 100.0% 73.3% Amorphous sulfasalazine154 372 372 367 30,300 136.5% 100.0% 25% sulfasalazine:HPMCAS-MG 34 725571 725 44,400 200.0% 146.5% 50% sulfasalazine:PVP VA64 67 1,372 1,2321,073 112,800 508.1% 372.3% 25% sulfasalazine:PVP VA64 425 2,350 2,3192,290 196,200 883.8% 647.5%

Example 10 Reformulation of Sulfasalazine Increases Oral BioavailabilityIn Vivo

The following experiments demonstrate that administration of a 25%sulfasalazine:PVP VA64 SDD composition results in a significant increasein oral bioavailability compared to administration of crystallinesulfasalazine in a rat model.

Preparation of Compounds

Crystalline sulfasalazine was obtained from Sigma-Aldrich (St. Louis,Mo.), Catalog # S0883. Crystalline sulfasalazine was re-suspended in0.5% Methocel (Methocel A4M Premium, Dow Chemical, Midland, Mich.) to aconcentration of 40 mg/ml sulfasalazine. Re-suspension of thecrystalline sulfasalazine composition was accomplished by adding the0.5% Methocel drop-wise to the composition and mixing in a mortar andpestle until the composition were evenly resuspended to form thenon-reformulated composition. Separately, a sample of 25%sulfasalazine:PVP VA64 SDD (Formulation Exemplar 2) was resuspended in0.5% Methocel to a concentration of 40 mg/ml sulfasalazine per ml. The25% sulfasalazine:PVP VA64 SDD composition was re-suspended by addingthe 0.5% Methocel drop-wise to the composition and mixing in a mortarand pestle until the composition were evenly resuspended, forming thereformulated composition.

Animal Study Design, Dosing and Plasma Collection:

A total of 6 Sprague-Dawley rats were used in the study, divided into 2cohorts as shown in Table 10. All rats were males that ranged in weightfrom 202 grams to 214 grams apiece. Rats were allowed to eat ad libitumbefore testing. Independently, the crystalline sulfasalazine formulationand the reformulated 25% sulfasalazine:75% PVP VA64 SDD composition wereadministered by gastric lavage at a dose of 400 mg/kg. Following drugadministration, 200 μl of plasma was collected from each animal at thefollowing time points: 30, 60, 90, 120, 160 and 240 minutes. Plasmasamples were snap frozen in liquid N₂ and stored at −80° C. untilanalysis.

Levels of sulfasalazine detected in the rat plasma at the different timepoints are given in Table 11, the summary and statistical values aregiven in Table 12 and the data presented in graphical format in FIG. 16.One rat (#6326) showed evidence that drug was partially administered tothe lungs, resulting in high plasma levels, and values from this ratwere not included in calculating mean values or in the statisticalanalysis. Oral administration of sulfasalazine, both the crystalline andthe 25% sulfasalazine:PVP VA64 SDD formulation showed immediate plasmaaccumulation within the first 30 minutes of administration. Thereformulated sulfasalazine (25% sulfasalazine:PVP VA64 SDD) showedhigher plasma levels, ranging from approximately 300% at the first 30minute time point to about 160% at the 3 hour time point, when comparedto the crystalline sulfasalazine composition following oraladministration.

TABLE 11 Concentrations of sulfasalazine in plasma. AnalyteSulfasalazine Levels, Plasma (ng/ml) Treatment Refomulation (25%GLX-1112:PVP-VA64 SDD) Parent (Crystalline) Subject ID 6326* 6327 6328Mean Levels 6329 6330 6331 Mean Levels Time 0.5 h 23,200 4,760 4,3704,565 1,390 1,270 1,800 1,487 Points 1 h 27,400 3,020 3,080 3,050 1,0501,380 991 1,140 1.5 h 17,100 2,980 2,910 2,945 1,340 1,810 1,130 1,427 2h 10,400 2,720 2,660 2,690 1,050 1,380 1,340 1,257 3 h 7,130 1,520 1,7801,650 939 787 1,360 1,029 4 h 3,790 271 1,050 661 872 790 887 850 *Testanimal exhibited evidence that drug was partially administered to lung;values for this animal were ommitted from mean value calculation.

TABLE 12 Summary and statistics of bioavailability experiment. Allstatistics were calculated using two-tailed Students t-test.Refomulation Percent (25% GLX- Parent difference: Time 1112:PVP-VA64(Crystalline) Reformulated/ Points SDD) Mean Values Mean Values Parentp-value 0.5 h 4,565 1,487 307% 0.0012 1 h 3,050 1,140 267% 0.0012 1.5 h2,945 1,427 206% 0.0100 2 h 2,690 1,257 214% 0.0018 3 h 1,650 1,029 160%0.0820 4 h 661 850  78% 0.7755

FIG. 16 shows the mean values of plasma sulfasalazine plotted ingraphical format.

The results of the above experiments demonstrate that: (1) thereformulated sulfasalazine attains higher plasma concentrationsfollowing oral administration than the crystalline formulation ofsulfasalazine and that (2) the increase in plasma concentrations areapproximately 300% percent to 160% over the first 3 hours ofadministration. These results demonstrate that sulfasalazine can bereformulated to increase oral bioavailability.

While a number of exemplary embodiments, aspects and variations havebeen provided herein, those of skill in the art will recognize certainmodifications, permutations, additions and combinations and certainsub-combinations of the embodiments, aspects and variations. It isintended that the following claims are interpreted to include all suchmodifications, permutations, additions and combinations and certainsub-combinations of the embodiments, aspects and variations are withintheir scope.

The entire disclosure of all documents cited throughout this applicationare incorporated herein by reference. All ranges set forth in thisspecification include the endpoints provided in those ranges unlessclearly indicated otherwise.

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What is claimed is:
 1. A method for treating a patient with aneurodegenerative disorder wherein the disorder is selected from thegroup consisting of seizures and glioblastoma, the method comprisingorally administering to the patient a pharmaceutical compositioncomprising a solid dispersion of sulfasalazine in essentially amorphousform and PVP VA64, wherein the ratio of sulfasalazine to PVP VA64 isbetween 20:80 wt./wt. and 30:70 wt./wt.
 2. The method of claim 1,wherein the solid dispersion is a spray-dried dispersion.
 3. The methodof claim 1, wherein the ratio of sulfasalazine to PVP VA64 is 25:75wt./wt.
 4. The method of claim 1, wherein the neurodegenerative disorderis seizures.
 5. The method of claim 1, wherein the neurodegenerativedisorder is glioblastoma.
 6. A method for treating a patient withseizures, the method comprising orally administering to the patient apharmaceutical composition comprising a solid dispersion ofsulfasalazine in essentially amorphous form and PVP VA64, wherein theratio of sulfasalazine to PVP VA64 is between 20:80 wt./wt. and 30:70wt./wt.
 7. The method of claim 6, wherein the solid dispersion is aspray-dried dispersion.
 8. The method of claim 6, wherein the ratio ofsulfasalazine to PVP VA64 is 25:75 wt./wt.
 9. A method for treating apatient with glioblastoma, the method comprising orally administering tothe patient a pharmaceutical composition comprising a solid dispersionof sulfasalazine in essentially amorphous form and PVP VA64, wherein theratio of sulfasalazine to PVP VA64 is between 20:80 wt./wt. and 30:70wt./wt.
 10. The method of claim 9, wherein the solid dispersion is aspray-dried dispersion.
 11. The method of claim 9, wherein the ratio ofsulfasalazine to PVP VA64 is 25:75 wt./wt.